generation of monoclonal antibodies against immunoglobulin...

Research ArticleGeneration of Monoclonal Antibodies against ImmunoglobulinProteins of the Domestic Ferret (Mustela putorius furo)

Greg A Kirchenbaum1 and TedM Ross12

1Center for Vaccines and Immunology University of Georgia Athens GA USA2Department of Infectious Diseases University of Georgia Athens GA USA

Correspondence should be addressed to Ted M Ross tedrossugaedu

Received 7 November 2016 Revised 20 December 2016 Accepted 12 January 2017 Published 14 February 2017

Academic Editor Kristen M Kahle

Copyright copy 2017 Greg A Kirchenbaum and Ted M Ross This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human diseasemost notably influenza Despite the importance of this animal model characterization of the immune response by flow cytometry(FCM) is severely hampered due to the limited number of commercially available reagents To begin to address this unmet needand to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells eight unique murinemonoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridomatechnology These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstratespecificity for CD79120573+ B cells Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enablesegregation of kappa and lambda B cells Additionally a mAb that yielded surface staining of nearly all surface BCR positivecells (ie pan ferret Ig) was generated Collectively these M120572F-Ig mAb offer advancement compared to the existing portfolioof polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including theidentification of antibody-secreting cells by FCM

1 Introduction

The domestic ferret (Mustela putorius furo) serves as ananimal model for the study of several viruses that causehuman disease [1 2] Most notably ferrets are naturallysusceptible to human influenza virus and are capable of viraltransmission [3ndash6] Their application to influenza researchbegan in 1933 when throat washings from human subjectswere administered intranasally into ferrets [6]These animalswent on to exhibit outward symptoms of influenza includingfever sneezing and lethargy Transmission of influenza-like disease was also observed following transfer of nasalwashings from an infected ferret or cohousing with a naıvecontact Due to the expression of both 12057226 and 12057223 sialicacid moieties along the respiratory tract ferrets are per-missive to infection with human seasonal and prepandemicavian influenza isolates [7] Moreover they recapitulate theextrapulmonary replication of highly virulent avian influenza

subtypes such as H5N1 and H7N7 [8 9] Collectively theferret model has provided invaluable insights into the anti-genicity virulence and transmissibility of circulating andnewly emerging influenza isolates [1 10ndash12]

Despite the usefulness of the ferret animalmodel the lackof ferret-specific reagents has severely hampered the abilityto perform in-depth immunologic profiling Recent studieshave implemented more detailed methods for interrogatingthe immune response elicited in the ferret including quanti-tative RT-PCR tomeasure cytokine and chemokine transcriptlevels and flow cytometric analysis of leukocyte populationsutilizing cross-reactive monoclonal antibody (mAb) reagents[13ndash17] However mAb with defined specificity for B celllineage surface markers such as CD19 [18] that would facili-tate identification of ferret B cells at various developmentalstages by flow cytometry (FCM) are currently lacking Tocircumvent this issue others have identified ferret B cellson the basis of surface immunoglobulin (Ig) andor CD79120572

HindawiJournal of Immunology ResearchVolume 2017 Article ID 5874572 13 pageshttpsdoiorg10115520175874572

2 Journal of Immunology Research

expression [14 16] While CD79120572 is an excellent marker foridentification of B lineage cells because it is an obligate chap-erone for surface expression of B cell receptor (BCR) [19]the epitope recognized by this mAb (clone HM47) requiresintracellular staining and thus does not permit isolation ofviable B cells [20] Alternatively polyclonal antibodies withreactivity against ferret Ig are conducive for surface stainingbut do not exclusively define B cells due to binding of ferretIg by myeloid cells via Fc receptors [14] Moreover neitherapproach enables segregation of ferret B cells on the basis ofheavy chain usage Collectively the suite of available reagentsis still insufficient for an in-depth characterization of thehumoral immune response and specifically the identificationof antibody-secreting cells by FCM To begin to address thisunmet need several novel mAb with specificity for ferretimmunoglobulin (Ig) were generated and characterized todefine their specificity

2 Materials and Methods

21 Animals BALBc mice (female 8ndash10 weeks of age) fromJackson Laboratory (Bar Harbor ME USA) and Fitch ferrets(Mustela putorius furo male or female 6 to 12 months ofage) from Triple F Farms (Sayre PA USA) were housed incage units and fed ad libitum All animals were handled inaccordance with protocols approved by Institutional AnimalCare and Use Committees and were cared for under USDAguidelines for laboratory animals

22 Isolation of Ferret Peripheral Mononuclear Cells Periph-eral blood was collected via cardiac puncture into vacuumcollection tubes containing sodium heparin (Becton Dickin-son Cat 367874) and gently inverted to prevent coagulationBlood was then combined with phosphate buffered saline(PBS) (Corning Cat 21-040-CV) to a final volume of 35mLand overlaid on 10mL Ficoll-Paque PLUS (GE HealthcareCat 17-1440) before centrifugation at 500timesg for 25minwith the brake reduced to its lowest setting using a SorvallLegend XTR (Thermo Scientific Grand Island NY USA)Peripheral blood mononuclear cells (PBMC) at the interfacewere collected washed with PBS and then pelleted (400timesgfor 10min) Following an additional wash with PBS totalcell number and viability was determined by Trypan blueexclusion using theCountess (ThermoFisher Cat C10227)Ferret PBMC were used immediately or resuspended in fetalbovine serum (FBS) (HyClone Cat SH3039603) containing10 DMSO (Thermo Scientific Cat 20688) for long-termstorage Aliquots of 1-2 times 107 viable ferret PBMC were storedin the vapor phase of liquid nitrogen until use and thawing ofcells was according to similarly described methods [22]

23 Purification of Ferret Immunoglobulin Serum from twoferrets (female 7 months of age) was pooled and immu-noglobulin (Ig) precipitated by drop-wise addition of anequal volume of saturated ammonium sulfate solution (41M)(Sigma Cat A4418) while maintaining the solution underconstant agitation at 4∘C Precipitated protein was pelletedby centrifugation at 115000 rpm for 20min at 4∘C and thendissolved in PBS The protein solution was then transferred

into a Slide-A-Lyzer dialysis cassette (ThermoFisher Cat66030) and dialyzed against PBS at 4∘C for three days withdaily buffer exchanges Subsequently the protein solutionwasclarified by centrifugation at 6000 rpm for 10min at 4∘C andthen passed through a 02 120583m syringe filter (ThermoFisherCat 09-719C) This material is referred to as crude ferretIg Ferret Ig was further purified by affinity chromatographyusing ProteinAG (ThermoFisher Cat 20423) Briefly crudeferret Ig protein solution was applied to Protein AG and thecolumn (Bio-Rad Cat 7311550) was washed by gravity flowwith PBS Fractions (2mL) were collected and absorbance(280 nm) was monitored using a PowerWave XS spectrom-eter (Biotek Winooski VT USA) Once wash fractionsreturned to baseline ferret Ig protein was eluted by additionof 01Mglycine pH 25 (Amresco Cat M103) Eluted proteinwas immediately neutralized with 200120583l 15M Tris pH88 (Amresco Cat M151) and protein containing elutionfractions were pooled buffer exchanged into PBS containing005 sodium azide (Sigma Cat S2002) and concentratedusing a Spin-XUF filter (Sigma Cat CLS431489)Thismate-rial is referred to as purified ferret Ig Protein concentrationsof the crude and purified ferret Ig solutions were determinedaccording to the manufacturersquos instructions using a microBCA assay kit (ThermoFisher Cat 23235)

24 ProteinGel Electrophoresis To assess purity of the respec-tive ferret Ig containing protein solutions 5120583g of crude orpurified ferret Ig was loaded into Bolt 10 Bis-Tris Plusprecast protein gels (ThermoFisher Cat NW00102) andresolved at 150V for 50min Protein samples were dilutedin either 2x Laemmli sample buffer (Bio-Rad Cat 161-0737)with or without 120573-mercaptoethanol (JT Baker Cat P62405)or 6x SDS-sample buffer (reducing) (Boston BioProductsCat BP-111R) Reduced samples were heated at 100∘C for10min and placed on ice prior to loading Gels were stainedwith PageBlue protein staining solution (ThermoFisherCat 24620) and then destained in water before imagingusing the myECL Imager (ThermoFisher Waltham MAUSA) Spectra Multicolor Broad Range Protein Ladder(ThermoFisher Cat 26634) was included in all gels and usedfor molecular marker reference

25 Generation of Murine Monoclonal Antibodies FemaleBALBc mice were immunized with 100 120583g of purified ferretIg containing the Imject alum adjuvant (Thermo ScientificCat 77161) via the intraperitoneal route Serumwas collectedvia the submandibular vein on day 21 and assessed by ELISAfor antibody reactivity (refer to ELISA subsection) The twomicewith the highest antibody titer received a booster immu-nization via the intraperitoneal route containing 100120583gof purified ferret Ig in PBS on Day 28 Three or fourdays after the booster immunization splenocytes wereharvested and used to perform a fusion with the SP2Omyeloma (kindly provided by Dr Lawrence Wysocki Uni-versity of Colorado Denver) using polyethylene glycol 1450(ATCC Cat 50-X) Hybridomas were selected by addi-tion of hypoxanthine (Acros Organics Cat 1220100) andazaserine (Sigma Cat A4142) at a final concentration of200120583M and 115 120583M respectively in RPMI 1640 (Sigma

Journal of Immunology Research 3

Cat R6504) containing 10 FBS 238mM sodium bicar-bonate (Fisher Scientific Cat BP328) 75mM HEPES(Amresco Cat 0485) 170 120583MPenicillin G (Tokyo ChemicalIndustry Cat P1770) 137 120583M Streptomycin (Sigma CatS9137) 50 120583M 120573-mercaptoethanol and 1mM sodium pyru-vate (ThermoFisher Cat 11360070) Eleven days after therespective fusions culture supernatants were screen forreactivity by ELISA (refer to ELISA subsection) Positivewellswere further expanded andmaintained under drug selectionand hybridoma cell lines of interest were subcloned by limit-ing dilution Hybridomas were subsequently expanded inmedia containing 5 IgG-stripped FBS (Hyclone CatSH3089803) and monoclonal antibody (mAb) purified byaffinity chromatography (Protein AG) as described above

26 ELISA The enzyme-linked immunosorbent assay(ELISA) was used to assess antibody reactivity against puri-fied ferret Ig and to determine the IgG subclass and concen-tration of the respective mAb To measure antibody bindingagainst the purified ferret Ig antigen CoStar high bindingELISA plates (Corning Cat 3590) were coated overnight at4∘C with 2 120583gmL purified ferret Ig in carbonate buffer pH94 containing 5 120583gmL fraction V bovine serum albumin(BSA) (Equitech-Bio Cat BAC69) Alternatively plates werecoated with 5 120583g BSA in carbonate buffer alone Plates wereblocked with ELISA block buffer PBS containing 02 BSA01 bovine gelatin (Sigma Cat G9391) and 005 Tween20 (Sigma Cat P7949) for 90min at 37∘C prior to addi-tion of culture supernatant or antibody solutions Culturesupernatants were diluted 1 2 for hybridoma screeningand purified mAb were diluted to 3 120583gmL in ELISA block-ing buffer prior to 3-fold serial dilution Plates were incu-bated for 90min at 37∘C and washed with PBS to removeunbound antibody For hybridoma screening horseradishperoxidase conjugated goat anti-mouse IgG (120574-specific)(Southern Biotech Cat 1030-05) secondary antibodydiluted in blocking buffer was added to ELISA plates Alter-natively binding of mAb to ferret Ig was revealed by additionof horseradish peroxidase conjugated goat anti-mouse IgG1(1205741-specific) (Southern Biotech Cat 1070-05) secondaryantibody After addition of secondary antibody plates wereincubated for 60min at 37∘C and then washed extensivelywith PBS prior to addition of 22-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS) (Amresco Cat 0400)substrate Plates were incubated at 37∘C for developmentand colorimetric conversion was terminated by addition of5 sodium dodecyl sulfate (SDS) (Teknova Cat S0294)Optical density was measured at 414 nm (OD414) using aPowerWave XS spectrophotometer

To determine the IgG subclass or determine the concen-tration of the respective mAb CoStar high binding ELISAplates were coated overnight at 4∘C with 1 120583gmL goatanti-mouse IgG (120574-specific) capture antibody (Sigma CatM1397) in carbonate buffer pH 94 containing 5120583gmL BSAPlates were then blocked with ELISA blocking buffer for90min at 37∘C Culture supernatants from clonal hybridomalines of interest diluted mouse anti-ferret Ig (M120572F-Ig) mAbor mouse IgG1120581 (Biolegend Cat 401402) were seriallydiluted in ELISA blocking buffer and plates incubated for

90min at 37∘C Plates were washed five times with PBShorseradish peroxidase conjugated goat anti-mouse IgG1(1205741-specific) secondary antibody diluted in ELISA blockingbuffer added and the plates incubated for 60min at 37∘CFollowing extensive washing with PBS ABTS substrate wasadded and plates incubated at 37∘C for development Col-orimetric conversion was terminated by addition of 5 SDSsolution and OD414 was measured using a PowerWave XSspectrophotometerThe concentration of individualmAbwasthen interpolated based on a nonlinear regression of theIgG1120581 standard using PRISM 60 (GraphPad Software LaJolla CA USA)

27 Competitive ELISA A competitive ELISAwas performedusing unlabeled and biotinylated M120572F-Ig mAb (refer to Pro-tein Conjugation) to identify overlapping epitope recogni-tion CoStar high binding ELISAplates were coated overnightat 4∘C with 2 120583gmL purified ferret Ig in carbonate buffer pH94 containing 5 120583gmL BSA Plates were then blocked withELISA blocking buffer for 90min at 37∘C Unlabeled M120572F-IgmAb (5ndash50120583gmL starting concentration determined usingBCA assay kit) were serially diluted in ELISA blocking bufferfollowed by addition of biotinylated M120572F-Ig mAb and platesincubated overnight at 4∘C Plates were washed five timeswith PBS horseradish peroxidase conjugated streptavidindiluted in blocking buffer added and the plates incubated for60min at 37∘C Following extensive washing with PBS ABTSsubstrate was added and plates incubated at 37∘C for devel-opment Colorimetric conversion was terminated by addi-tion of 5 SDS solution and OD414 was measured using aPowerWave XS spectrophotometer The percent of maximalsignal was determined using the formula 100 times [OD414experimental sample minus OD414 blankOD414 maximal signalminus OD414 blank]

28Western Blot To characterize the specificity of individualM120572F-IgmAb 1120583g of purified ferret Ig or 05120583l of ferret serumwas reduced and resolved by protein gel electrophoresis asdescribe previously Protein transfer to polyvinylidene difluo-ride (PVDF)membranes was performed using the Trans-BlotTurbo RTA Mini PVDF transfer kit (Bio-Rad Cat 1704272)and a Trans-Blot Turbo Blotting system (Bio-Rad HerculesCA USA) according to the manufacturersquos instructions Themembrane was blocked with PBS + Tween 20 (01 vv)(PBST) containing 5 BSA (VWR Cat 0332) at room tem-perature (RT) with constant agitationThe PVDFmembraneswere then cut into strips and probed with 15mL of PBSTcontaining 01 120583gmL of individual M120572F-Ig mAb overnight atRT The following day PVDF membranes were washed threetimes with PBST before incubation for 60min at RT with10mL PBST containing horseradish peroxidase conjugatedgoat anti-mouse IgG1 (1205741-specific) Following extensivewash-ing with PBS membranes were treated with 4mL ClarityWestern ECL Substrate (Bio-Rad Cat 1705060) and imagedusingmyECL Imager Postacquisition analysiswas performedusing myImageAnalysis Software (ThermoFisher)

29 Flow Cytometry To evaluate the specificity of com-mercially available anti-ferret Ig reagents by flow cytometry


(FCM) ferret PBMC were labeled with FITC conjugatedgoat anti-ferret IgM (120583-specific) (Sigma Cat SAB3700807)or biotin conjugated goat anti-ferret IgG (G120572F-Ig120574 BIO)(Sigma Cat SAB3700796) Binding of G120572F-Ig120574 BIO wasrevealed using phycoerythrin conjugated streptavidin (SA-PE) (Biolegend Cat 405204) The reactivity of individualmAb against ferret leukocytes was assessed by direct orindirect staining Initially culture supernatants from clonalhybridoma lines were diluted to 1 120583gmL in FCM stainingbuffer (PBS + 2 FBS + 01 NaN3) and used to stain ferretPBMC Because this screening method was dependent onrevealing binding of the murine mAb using a polyclonalgoat anti-mouse Ig Alexa Fluor 647 (G120572M-Ig AF647) reagent(BD Cat 51-9006588BK) we were unable to utilize a cross-reactive anti-CD79120573murinemAb (clone CB3-1) to identify Bcells [16 23] Ferret B cells were instead identified using G120572F-Ig120574 BIO which was revealed using SA-PE Binding of murinemAb to ferret PBMC was revealed with G120572M-Ig AF647 Allstaining was performed on ice in 100120583l of volume and cellswere stained with LIVEDEAD Aqua (ThermoFisher CatL34957) prior to surface staining to enable exclusion ofnonviable cells

For direct staining ferret PBMC were initially stainedwith LIVEDEAD Aqua and then pretreated with irrelevantMouse IgG2a120581 (Biolegend Cat 401502) and Rat IgG2a120581(Biolegend Cat 400502) to exclude nonviable cells andmin-imize nonspecific binding Ferret PBMC were then stainedwith anti-CD79120573 (Biolegend Cat 341408) and DyLight 488DyLight 650 andor biotin conjugated M120572F-Ig mAb (referto Protein Conjugation) Biotinylated mAb were revealed bysecondary stainingwith SA-PE In order to putatively identifykappa light chain expressing B cells ferret PBMC werestained with DyLight 650 conjugated recombinant Protein L(ThermoFisher Cat 21189) and anti-CD79120573 simultaneouslywith DyLight 488 and biotin conjugated M120572F-Ig mAbAcquisition was performed on the LSR II cytometer (BDBiosciences San Jose CA USA) and analysis performedwithFlowJo Version 1008 (Tree Star Ashland OR USA)

210 Protein Conjugation Protein AG purified mAb orrecombinant Protein L were conjugated to DyLight 488(ThermoFisher Cat 46402) DyLight 650 (ThermoFisherCat 62265) or EZ-Link NHS-LC-Biotin (ThermoFisherCat 21336) according to the manufacturersquos instructionsUnreacted DyLight or NHS-LC-Biotin was removed bymultiple buffer exchanges into PBS containing 005 sodiumazide using Spin-X UF filters (30 kDa MWCO)

211 Sequencing Hybridoma V Region Genes Variable genesof B cell hybridomas were cloned by RT-PCR and 51015840 rapidamplification of cDNA ends (RACE) using pairs of constantregion and anchor primers according to previously describedmethods with minor modifications [24] Briefly total RNAwas isolated from hybridoma lines using RNeasy (QiagenCat 74104) and first-strand cDNA synthesis performedusing SuperScript III RT (ThermoFisher Cat 18080051)using oligo-dT primer First-strand cDNA was isolated usingQIAquik PCR spin columns (Qiagen Cat 28104) andthen dG-tailed with TdT (NEB Cat M0315S) and dGTP

(ThermoFisher Cat 10218014) Variable IgH or Ig120581 geneswere then amplified from dG-tailed cDNA templates usingPhusion (NEB Cat M0530S) A poly-A tail was addedto products following completion of the second round ofPCR by addition of 5 units recombinant Taq polymerase(ThermoFisher Cat EP0402) directly into the reaction andincubation at 72∘C for 10min Products from Ig120581 PCR werefurther purified with QIAquick PCR spin columns beforedigestion with restriction enzymes PflFI (NEB Cat R0595S)or PflmI (NEB Cat R0509S) to disrupt the rearrangedV12058121-12 gene from the SP20 fusion partner After 2 agaroseelectrophoresis the uncut V120581 products were isolated usingthe QIAquik gel extraction kit (Qiagen Cat 28704) andeluted with autoclaved water Variable region genes werecloned into pCR4-TOPO (ThermoFisher Cat K4575J10) orpSC-A (Agilent Cat 240205) plasmids according to themanufacturersquos instructions Plasmid DNA was purified usingQIAprep spin columns (Qiagen Cat 27104) and submittedto Macrogen (Rockville MD USA) for sequencing Heavyand kappa variable region genes were identified using IMGTV-Quest [21]

212 Statistics Statistical analyses were performed usingPRISM 60

3 Results

31 Commercial Reagents against Ferret Immunoglobulin LackHeavy Chain Specificity Expression of a class-switched B cellreceptor (BCR) such as IgG or IgA can be used as a markerof memory B cells while naıve B cells express an IgM BCR[25] As a first attempt to segregate ferret B cells into naıve andmemory compartments on the basis of surface BCR expres-sion ferret PBMCwere stained with polyclonal goat anti-fer-ret IgM (G120572F-Ig120583) or goat anti-ferret IgG (G120572F-Ig120574) antise-rum Additionally the mouse anti-human CD79120573 mAb(cloneCB3-1) which cross-reacts with ferret leukocytes (Sup-plementary Materials available online at httpsdoiorg10115520175874572) was included in the staining solutionto identify surface BCR+ cells [16] The G120572F-Ig120583 antiseralabeled sim99 of the CD79120573+ population while the G120572F-Ig120574antisera costained sim66 of the CD79120573+ population (Supple-mentary Materials) To extend these observations ferretPBMCwere next stainedwith both the G120572F-Ig120583 andG120572F-Ig120574simultaneouslyThemajority of CD79120573+ ferret PBMC exhib-ited staining with both the G120572F-Ig120583 and G120572F-Ig120574 reagentsCollectively these findings indicate that surface stainingwith anti-CD79120573 enables identification of ferret B cells andcurrently available anti-ferret Ig reagents are insufficient todiscriminate B cells on the basis of heavy chain expression

32 Purification of Ferret Immunoglobulin Ferret Ig was firstcrudely enriched from serum through ammonium sulfateprecipitation and the resulting protein solution was predomi-nantly IgG (Figure 1 lanes 2 and 3) Next ferret Ig was furtherpurified by affinity chromatography using Protein AG Thissecond purification step removed the majority of contami-nating proteins and produced a highly pure ferret Ig prepara-tion (Figure 1 lanes 4 and 5) Reduction of the purified ferret


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Figure 1 SDS-polyacrylamide gel electrophoresis of purified ferretimmunoglobulin 5120583g of crude (lanes 2 and 3) or Protein AGpurified (lanes 4 and 5) ferret Ig preparations were resolved by SDS-PAGE under nonreducing (lanes 2 and 4) or reducing (lanes 3 and5) conditions Molecular marker references are indicated (lane 1)Image was acquired using the myECL imager and contrast adjust-ment of the entire image was performed using myImageAnalysissoftware Image is representative of two independent purifications

Ig into heavy and light chain components confirmed thepresence of both Ig120583 and Ig120574 on the basis of their differentialsizes (Figure 1 lane 5) [26]

33 Immunization with Purified Ferret ImmunoglobulinMouse IgG2a mAb have increased nonspecific binding toferret leukocytes relative to IgG1 (data not shown) In orderto elicit an antibody response utilizing the IgG1 subclassBALBc mice were immunized with purified ferret Ig pre-pared with the Imject alum adjuvant [27] Following asingle immunization with ferret Ig and adjuvant antigen-specific reactivity was detected by ELISA and western blot(Supplementary Materials) Moreover reduction of ferret Igin the SDS-PAGE enabled discrimination between antibodyreactivity with the light (Ig120581Ig120582) and heavy (Ig120583 or Ig120574) chaincomponents of ferret Ig

34 Characterization of Monoclonal Antibodies by ELISABased on their reactivity with ferret Ig two mice (3 and 5)were chosen for mAb generation Eight IgG1+ mAb collectedfrom two independent fusions reacted with purified ferretIg Each of these mouse anti-ferret Ig (M120572F-Ig) mAb reactedwith ferret Ig by ELISA (Figure 2) Additionally normaliza-tion of the input IgG1 concentration highlighted the distinctbinding curves of several M120572F-Ig mAb

35 Assessment of Monoclonal Antibody Reactivity byWesternBlot As an additional technique to further characterize thereactivity of the respective mAb with ferret Ig individualM120572F-Ig mAb were used to probe reduced ferret Ig antigenand serum from three naıve ferrets in a western blot screen

Ferret Ig

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Figure 2 Monoclonal antibody binding to ferret immunoglobulinMouse mAb were evaluated for binding to purified ferret Ig byELISA Optical densities are plotted versus normalized IgG1 input(119909-axis) Data are representative of two or more independentexperiments that yielded similar results

Three of the eight M120572F-Ig mAb demonstrated specific reac-tivity with ferret Ig antigen using this assay (Figure 3) BothM120572F-Ig mAb 4-B10 and 8-H9 reacted with ferret Ig lightchain protein (Figures 3(b) and 3(c)) Additionally mAb 11-E3 reacted with a protein species corresponding to the Ig120574chain (Figure 3(d)) Of note these M120572F-Ig mAb reacted withboth purified ferret Ig antigen and serum samples suggest-ing the epitopes recognized by the respective mAb are notpolymorphic

36 Assessment of Monoclonal Antibody Reactivity by FlowCytometry In spite of earlier observations suggesting thatthe G120572F-Ig120574 antisera was unlikely to define IgG+ ferretB cells exclusively (Supplementary Materials) this reagentwas incorporated into a flow cytometric screening assay toevaluate the ability of eachM120572F-Ig mAb to bind ferret PBMCbecause it enabled resolution of these cells into distinct pop-ulations on the basis of staining intensity (Figure 4(b))The two populations of ferret PBMC that demonstratedstaining with the G120572F-Ig120574 antisera are referred to as IgGint

and IgGhi respectively for the sole purpose of detailing thestaining patterns observed for the respective M120572F-Ig mAb(Figure 4 and data not shown) Using this flow cytometricscreening approach both 4-B10 and 8-H9had similar stainingpatterns of ferret PBMC and labeled sim50 of the IgGhi

population (Figures 4(c) and 4(d)) Moreover both of theseM120572F-Ig mAb also reacted with a small population of IgGint

cells Three additional M120572F-Ig mAb (4-E3 4-D11 and 6-H5)had similar reactivity patterns (data not shown) A distinctstaining pattern was observed using two M120572F-Ig mAb (6-B5 and 6-B7) (Figure 4(e) and data not shown) Specifi-cally pretreatment of ferret PBMC with these mAb resulted


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Figure 3 Reactivity of monoclonal antibody against reduced ferret immunoglobulin 1120583g of ferret Ig (lane 2) or 05 120583l of ferret serum (119899 = 3)(lanes 3ndash5) was resolved by SDS-PAGE under reducing conditions and transferred to PVDFmembranesMembranes were then cut into stripsand probed with (a) irrelevant IgG1 (b) 4-B10 (c) 8-H9 or (d) 11-E3 Images were acquired using themyECL imager and contrast adjustmentsof the entire image were performed using myImageAnalysis software Visible light images of the molecular markers (lane 1) are presentednext to the corresponding chemiluminescent image for reference Images are representative of two independent experiments

in the disappearance of the IgGhi population likely due toBCR internalization or blocking of epitopes targeted by theG120572F-Ig120574 antisera Additionally a subset of the IgGint popu-lation was labeled by 6-B7 on the basis of G120572F-Ig120574 staining(Figure 4(e)) Finally the 11-E3 mAb had a third stainingpattern with low-level binding to IgGint cells but did not bindto IgGhi cell population

As a next step to further characterize the reagents directlylabeledM120572F-Ig mAb were used to stain ferret PBMC in com-bination with anti-CD79120573 (Figure 5) Based on previouslyobserved western blot and flow cytometric staining patterns(Figures 3 and 4) the M120572F-Ig mAb were segregated into twoclassifications The first group of M120572F-Ig mAb comprised 6-B5 6-B7 and 11-E3 and these mAb had a reactivity profileconsistent with Ig heavy chain specificity Despite evidenceof reactivity with IgGhi cells in the indirect screen (data notshown) biotinylated 6-B5 did not stain CD79120573+ ferret B cells(Figure 5(a)) By contrast biotinylated 6-B7 stained almost allof the CD79120573+ cell population (Figure 5(b)) In addition 11-E3 stained the entire CD79120573+ cell population with a low-levelof fluorescence as well as a small population of CD79120573negcells (Figure 5(c)) Similar results were also observed usingthese same mAb following DyLight conjugation (data notshown)

The second group of M120572F-Ig mAb (4-B10 and 8-H9)exhibited a reactivity profile consistent with light chainspecificity However double-labeling CD 79120573+ ferret PBMCwith these mAb revealed staining of distinct populationsof cells (Figure 6(a)) Moreover nearly the entire CD79120573+population stained positive with either 4-B10 or 8-H9 withfew cells reacting with both mAb Collectively these findingssuggested that 4-B10 and 8-H9 define distinct populations offerret B cells on the basis of light chain expression

The remainingM120572F-Ig mAb (4-E3 4-D11 and 6-H5) alsohad light chain reactivityThesemAbwere categorized on thebasis of costaining with 4-B10 or 8-H9 single-positive ferretB cells (Figures 6(b)ndash6(d)) Using a multilabeling approach4-E3 was found to costain with nearly the entire 4-B10pospopulation while simultaneously not labeling 8-H9pos cells(Figure 6(b) and data not shown) Similarly 8-H9pos cellswere labeled by 4-D11 or 6-H5 and these mAb exhibitedminimal reactivity with 4-B10pos cells (Figures 6(c) and 6(d))Of note we routinely observed a small population of 8-H9pos cells that were not stained by 4-D11 (Figure 6(c))Collectively these data indicate that 4-B10 and 4-E3 recognizea common light chain protein that is distinct from the lightchain recognized by the other mouse anti-ferret light chain(M120572F-IgL) mAb (8-H9 4-D11 and 6-H5)

37 Identification of Overlapping Epitope Recognition throughCompetitive Binding ELISA To further characterize whetherthe M120572F-Ig mAb were recognizing overlapping or distinctepitopes on ferret Ig competitive binding assays were per-formed Specifically unlabeled M120572F-Ig mAb were used ascompetitors and evaluated for their ability to inhibit bindingof biotinylated mAb (4-B10 4-E3 6-B7 8-H9 and 11-E3) tothe purified ferret Ig antigen Consistent with the double-labeling FCM studies in which 4-B10 and 8-H9 recognizeddistinct populations of ferret PBMC (Figure 6(a)) theseM120572F-Ig mAb did not exhibit inhibition of each other in thecompetitive binding ELISA (Figures 7(a) and 7(c)) In addi-tion despite recognition of the same population as assessedby double-labeling FCM (Figure 6(b)) 4-E3 exhibited onlysubtle inhibition of 4-B10 at the highest concentration tested(5 120583gmL) (Figure 7(a)) In stark contrast 4-B10 competedfor binding with biotinylated 4-E3 (Figure 7(b)) Moreover


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or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)

Figure 4 Flow cytometric assessment of monoclonal antibody reactivity Reactivity of mouse anti-ferret Ig mAb with ferret PBMC wasevaluated by flow cytometry Binding of mouse Ig to ferret PBMC was revealed by secondary staining with Alexa Fluor 647 conjugatedgoat anti-mouse IgG (G120572M-IgG) Additionally ferret B cells were identified by costaining with biotinylated goat anti-ferret IgG which wasrevealed by secondary staining with phycoerythrin conjugated streptavidin (SA-PE) (a) Reactivity of the G120572M-IgG secondary antibody withferret PBMC (bndashf) 1120583g of an IgG1 control (b) or mouse anti-ferret Ig mAb (cndashf) was used for indirect surface staining of ferret PBMC Thepresented data were generated using PBMC from a single ferret and are representative of two or more independent experiments that yieldedsimilar results

mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)

Figure 5 Surface staining with heavy chain reactivemonoclonal antibodies Biotinylatedmouse anti-ferret IgmAbwere used in combinationwith anti-CD79120573 to evaluate surface staining of ferret B cells Binding of biotinylated (a) 6-B5 (b) 6-B7 or (c) 11-E3was revealed by secondarystaining with phycoerythrin conjugated streptavidin (SA-PE) Frequency of CD79120573negpos ferret PBMC that costained with the mouse anti-ferret Ig mAb are indicated Data shown were generated using a single ferret and are representative of two or more independent experimentsthat yielded similar results


mAb 4-B10 (DyLight 488)

mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

mAb 8-H9 (DyLight 650)

mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)

Figure 6 Characterization of light chain reactive monoclonal antibodies Mouse anti-ferret Ig mAb were used in combination for surfacestaining of ferret PBMC Plots are pregated on B cell receptor expressing cells (CD79120573+) as shown in Supplementary Materials (a) Dualstaining of ferret B cells with 4-B10 and 8-H9 (b) Dual staining of ferret B cells with 4-B10 and 4-E3 (c) Dual staining of ferret B cells with8-H9 and 4-D11 Arrow indicates population of 8-H9+ B cells that lack costaining with 4-D11 (d) Dual staining of ferret B cells with 8-H9 and6-H5 Data are representative of two or more independent experiments comprising 119899 ge 6 individual ferrets

4-B10 exhibited superior competition with biotinylated 4-E3 relative to the homologous unlabeled competitor Nocompetition of biotinylated 8-H9 was observed by any M120572F-Ig mAb tested Strikingly even unlabeled 4-D11 at 20 120583gmLfailed to compete with 8-H9 despite these mAb labeling acommon population of ferret PBMC by FCM (Figures 6(c)and 7(c)) Similarly none of the M120572F-Ig mAb exhibitedcompetition with biotinylated 11-E3 (Figure 7(d)) Howevera number of M120572F-IgL mAb were found to compete withbiotinylated 6-B7 despite assignment of this mAb as heavychain specific (Figure 7(e)) Specifically both unlabeled 4-B10and 8-H9 demonstrate definitive competition with biotiny-lated 6-B7 (Figure 7(e)) Moreover 4-E3 and 4-D11 (only athigh concentrations) were also capable of inhibiting binding

of biotinylated 6-B7 to the ferret Ig antigen albeit to a lessextent relative to 4-B10 and 8-H9

38 Determination of Light Chain Specificity Although theindividual M120572F-IgL mAb were already segregated into twodistinct groups on the basis of light chain reactivity theirprecise specificity remained undefined To determine whichgroup of M120572F-IgL mAb was specific for the Ig120581 chain afluorescently labeled bacterial protein with specificity for Ig120581chains from a variety of species [28 29] Protein L was usedto label ferret B cells Approximately 25 of CD79120573+ ferretB cells stained brightly with the fluorescently conjugatedProtein L reagent (Figure 7(a)) and these cells were putativelyassigned as Ig120581-expressing B cells Next these CD79120573+


4-B10 (5 ngwell)

Concentration (ngmL)

98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal

IgG1휅 isotype4-B10 (IgL)4-E3 (IgL)8-H9 (IgL)

6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al

IgG1휅 isotype4-B10 (IgL)4-D11 (IgL)8-H9 (IgL)

6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al

IgG1휅 isotype4-B10 (IgL)4-E3 (IgL)4-D11 (IgL)

8-H9 (IgL)6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(e)

Figure 7 Mapping of monoclonal antibodies through competitive ELISA Inhibition of biotinylated mouse anti-ferret Ig mAb binding topurified ferret Ig was performed to identify overlapping epitope recognition Competitive inhibition of (a) 4-B10 (b) 4-E3 (c) 8-H9 (d)11-E3 and (e) 6-B7 were performed using the indicated concentration of biotinylated mAb The 119909-axis indicates concentration of unlabeledmouse anti-ferret Ig mAb competitor and the homologous unlabeled mAb are depicted in red Percent of maximal binding by the respectivebiotinylated mAb is plotted plusmn SD of duplicates Data in (a)ndash(e) are representative of two independent experiments

Protein Lhi cells were evaluated for costaining with 4-B10 or8-H9 which defined distinct subsets of ferret B cells Themajority (sim80) of CD79120573+ Protein Lhi cells were costainedwith 4-B10 (Figure 7(b)) and indicated that 4-B10was specificfor ferret Ig120581 Conversely this result indicated that 8-H9 wasspecific for Ig120582 Using these assignments the distribution ofIg120581 and Ig120582 expression by circulating CD79120573+ ferret B cellswas determined (Figure 7(c))

4 Discussion

In this study eight individual mAb with specificity for ferretIg were characterized (Table 1) Five of these mAb hadspecificity for ferret Ig light chain while the remaining threemAb did not recognize a distinct surface expressed heavychain Additionally we generated a mAb (6-B7) that yieldedsurface staining of nearly all surface BCR positive cells(ie pan ferret Ig) Collectively these M120572F-Ig mAb offeradvancement in ferret Ig detection compared to the existing

portfolio of polyclonal antisera reagents Moreover severalof these mAb (4-B10 8-H9 and 11-E3) are likely to beapplicable to a wide array of immunologic assays includingthe identification of antibody-secreting cells by FCM

Pooled serum was chosen as the source of material forsubsequent purification of ferret Ig because both IgM andIgG antibody classes were abundant and this biological fluidwas readily available [26] Moreover the purified ferret Igpreparation had the potential to elicit mAb with specificityfor the heavy chain (Ig120583 or Ig120574) and the light chain (Ig120581 orIg120582) using a single antigen However due to the complexityof the ferret Ig antigen antigen-specific binding in the ELISAformat was insufficient to determine the precise specificity ofthe respectiveM120572F-IgmAb (Figure 2) To this end additionalscreening approaches such as FCM western blot and com-petitive binding assays were necessary to further define thespecificity and categorized the individual M120572F-Ig mAb

Implementation of an indirect FCM screening methodduring the initial characterization enabled identification of


Prot

ein

L D

yLig

ht 6

50

CD79훽 PerCP-Cy55 mAb 4-B10 DyLight 488(b) (c)(a)

mAb

8-H

9 BI

O +

SA-

PE Light chain usage

Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast

Figure 8 Designation of light chain reactive monoclonal antibody specificity Mouse anti-ferret Ig mAb (4-B10 and 8-H9) were used incombination with anti-CD79120573 and DyLight 650 conjugated Protein L to assign mAb reactivity with kappa light chain expressing B cells (a)Representative surface staining of ferret PBMCwith anti-CD79120573 and Protein L Kappa light chain expressing B cells were defined as CD79120573+cells that stained intensely with DyLight 650 conjugated Protein L and were gated as shown in (a) The mean frequency of total B cells plusmn SDthat stained intensely with Protein L is indicated (b) Representative costaining of kappa light chain expressing B cells with 4-B10 or 8-H9The mean frequency plusmn SD of total kappa B cells exhibiting costaining with 4-B10 or 8-H9 is indicated Data in (a) and (b) are combined fromtwo independent experiments (119899 = 6) (c) Distribution of kappa or lambda light chain expression by total B cells (CD79120573+) was determinedon the basis of costaining with 4-10 or 8-H9 Data in (c) are combined from three independent experiments (119899 = 14) Statistical significancewas determined using a paired Studentrsquos 119905-test lowastlowastlowast119901 lt 0001

Table 1 Summary of M120572F-Ig mAb

mAb Ig subclass VHa VL

a Specificity4-B10 IgG1 IGHV4-1 IGKV6-13 Light chain (Ig120581)cd

4-D11 IgG1 IGHV2-3 IGKV16-104 Light chain (Ig120582)d

4-E3b IgG1 IGHV1-20 or IGHV1-37 IGKV3-12 Light chain (Ig120581)d

6-B5 IgG1 IGHV5-6 IGKV19-93 Ferret Ig6-B7 IgG1 IGHV5-6 NDef Heavy chain (IgH)g

6-H5 IgG1 IGHV5-17 IGKV5-48 Light chain (Ig120582)d

8-H9 IgG1 IGHV14-3 IGKV6-32 Light chain (Ig120582)cd

11-E3 IgG1 IGHV1S81 IGKV6-17 Heavy chain (Ig120574)c

a identified using IMGT V-Quest (see [21]) b unable to unambiguously assign IGHV c determined by western blot d determined by flow cytometry e notdetermined f negative for intracellular Ig120581 by flow cytometry g determined by competitive ELISA

M120572F-Ig mAb that recognized epitopes present on ferretleukocytes Moreover incorporation of the G120572F-Ig120574 antiserain this screen enabled resolution of ferret PBMC into distinctpopulations on the basis of staining intensity and providedinsight into the specificity of the respective M120572F-Ig mAb(Figure 4 and data not shown) Specifically each of M120572F-IgLmAb exhibited a similar staining pattern in which approx-imately half of the sIgGhi population was intensely labeledby the respective mAb Moreover these M120572F-IgL mAb alsolabeled a small subset of the sIgGint population at a similarlyintense level while the remainder of the sIgGint populationexhibited a low-level of staining (Figures 4(c) and 4(d) anddata not shown) This staining pattern indicated that surfaceIg+ B cells constituted a fraction of the sIgGint populationand was consistent with the observation that the G120572F-Ig120574antisera as well as other commercially available polyclonalanti-ferret Ig antisera fail to label all B cells (data not shownand [14]) Additionally this staining pattern implied thatmyeloid cells constituted a portion of the sIgGint populationand that IgG acquired via FcR was bound in such manner

that the light chain was accessible to the respective M120572F-IgL mAb Furthermore segregation of the IgGint and sIgGhi

populations in the FCM screen also facilitated perception ofthe distinct binding specificities exhibited by the heavy chainreactive mAb 6-B7 and 11-E3 on the basis of their differentialreactivity profile with the sIgGhi population (Figures 4(e) and4(f)) Lastly in the absence of the apparent disappearance ofthe sIgGhi population that resulted following pretreatment offerret PBMC with 6-B5 (data not shown) this M120572F-Ig mAbwould not have been characterized further

Despite the perceived reactivity of 6-B5 in the antigen-specific capture ELISA and indirect FCM screening duringthe initial characterization definitive staining of ferret B cellsusing this mAb when directly coupled was not observed(Figure 5(a) and data not shown) It is likely that biotiny-lation of 6-B5 was inefficient rather than that conjugationdestroyed the binding specificity of this mAb Specificallyboth unlabeled and biotinylated 6-B5 exhibited comparablebinding to ferret Ig in an ELISAwhen using a goat anti-mouseIgG1 secondary reagent (data not shown) By contrast the


same biotinylated 6-B5 exhibited a near absence of reactivityagainst ferret Ig when streptavidinwas used to reveal bindingConsequently it will be necessary to explore alternativechemistries for conjugation of 6-B5 to fully realize the utilityof this mAb for flow cytometric applications

The competitive binding ELISA contributed to the char-acterization of the respective M120572F-Ig mAb First the datagenerated using this approach served to reaffirm priorobservations such as the distinction between 4-B10 and 8-H9 determined by double-labeling FCM Second the com-petitive binding and antigen-specific capture ELISA datawere in agreement and reflected differences in functionalaffinity by several of the M120572F-Ig mAb Most notably 4-B10exhibited stronger avidity for the ferret Ig antigen relative to4-E3 (Figure 2) and also demonstrated superior competitionof biotinylated 4-E3 compared to the homologous mAb(Figure 7(b)) Thirdly the competitive binding ELISA alsoprovided additional insight into the epitopes recognized bythe respective M120572F-Ig mAb While it was already presumedthat the M120572F-IgL mAb were targeting the constant regionof kappa or lambda light chain based on their FCM stainingpatterns (Figure 6) it remained unclear if these anti-kappa oranti-lambda mAb were targeting distinct or overlapping epi-topes On the basis of competitive inhibition it is likely that4-B10 and 4-E3 are recognizing similar but not completelyoverlapping epitopes on the kappa constant region since theinhibition was unidirectional (Figures 7(a) and 7(b)) Bycontrast 4-D11 failed to inhibit binding of biotinylated 8-H9 despite the use of an elevated concentration (20120583gmL)suggesting that these mAb recognize distinct epitopes onthe lambda constant region However it remains plausiblethat the large difference in functional affinity between thesemAb contributed to the lack of competition (Figures 2 and7(c)) Additionally biotinylated 4-D11 failed to produce asufficiently high binding signal against the ferret Ig antigen toenable evaluation of reciprocal inhibition by 8-H9 (data notshown) Similar to 8-H9 no inhibition of biotinylated 11-E3was observed using any of the M120572F-Ig mAb tested furthersupporting the notion that thismAb recognizes a distinct epi-tope (Figure 7(d)) Surprisingly several of the M120572F-IgL mAbwere capable of inhibiting binding of biotinylated 6-B7 to theferret Ig antigen This was unexpected since 6-B7 was cate-gorized as heavy chain reactive based on the observed FCMstaining pattern (Figure 5(c)) However the ability of 4-B10and 8-H9 aswell as 4-E3 and 4-D11 to a less extent to competewith 6-B7 supports the conclusions that 6-B7 is recognizingan epitope that is restricted to the heavy chain Moreoverthese data suggest that 6-B7 recognizes an epitope on theheavy chain of ferret Ig that is proximal to either the kappa orlambda constant region To this endwe hypothesize that 6-B7would maintain reactivity with a Fab fragment of ferret Ig

In spite of strong reactivity with purified ferret Ig byELISA (Figure 2) and the Ig120574 chain by western blot (Fig-ure 3(d)) intense staining of ferret B cells using directlylabeled 11-E3 was not observed (Figure 5(c)) However low-level staining of the entire CD79120573+ B cell population aswell as a small population of non-B cells in bulk ferretPBMC was observed using 11-E3 Collectively these obser-vations indicate that the epitope recognized by 11-E3 may be

restricted to secreted ferret IgG Further experimentationwillbe necessary to resolve the utility of 11-E3 for studying ferretB cells and derived Ig

The assignment of lambda light chain specificity to 8-H9 was based upon several independent observations Firstthe near absolute segregation between populations of 4-B10+and 8-H9+ ferret B cells indicated that these mAb recog-nized fundamentally distinct light chains (Figure 6(a)) Ofnote this observation also indicates that allelic exclusionof dual light chain expression is largely intact in the ferretmodel [30] Secondly 8-H9+ ferret B cells were severelyunderrepresented in the population of B cells that bound thekappa-specific Protein L reagent at high levels (Figures 8(a)and 8(b)) While there was a population of 8-H9+ B cellsin this gate (sim20) it is plausible that these Ig120582+ B cellsacquired labeling with Protein L either due to inherent BCRspecificity or through the association of secreted Ig120581 withsurface expressed FcR [31 32] In addition within the total Bcell population the frequency of 8-H9+ B cells detected frommultiple independent ferrets closely resembled the distribu-tion of Ig120582+ B cells observed in humans [33]

While a draft sequence of the ferret genome is currentlyavailable [34] the Ig loci have not been annotated Based onthe findings presented in this report we hypothesize that theferret Ig120582 locus more closely resembles the human Ig120582 locusthan the mouse Ig120582 loci due to the increase proportion ofB cells that express a lambda light chain (Figure 8(c)) Inlaboratory mice the Ig120582 locus is comprised of 3 functionalV120582 gene elements that rearrange with associated J120582-C120582 geneelements [35] Additionally in mice the prevalence of serumimmunoglobulin utilizing a lambda light chain is severelyreduced relative to immunoglobulin utilizing a kappa lightchain [36] By contrast the human Ig120582 locus is more complexand encodes greater than 35V120582 genes belonging to 10 sub-groups [37] Moreover the distribution of kappa and lambdaimmunoglobulin is more balanced in human serum [38]Collectively these findings provide the first evidence thatlambda light chain usage in ferrets closely resembles that seenin humans and further supports the use of ferrets for studyingthe antibody response elicited by influenza virus infection orinfluenza vaccination

To more accurately mimic influenza infection and vac-cination in humans ferrets can be infected with variousinfluenza viruses fromdifferent subtypes to establish a preim-mune state in the animal This model is useful to study bothinactivated and live attenuated influenza vaccine (LAIV) can-didates In young children an IgA recall response occurredfollowing a second administration of LAIV [39] Howeverferrets previously infected with LAIV had a robust IgGantibody-secreting cell (ASC) response in the absence of anaccompanying IgA ASC population following experimentalchallengewith seasonal influenza [40]While it remains plau-sible that the immune response elicited by influenza infectionof preimmune ferrets was distinct from that observed inyoung children following LAIV inoculation it is more likelythat the discrepancy between these two models originatesfrom the use of polyclonal anti-ferret Ig detection reagentsthat possess an inherent lack of heavy chain specificityThis apparent lack of heavy chain specificity was observed


using a variety of polyclonal goat anti-ferret Ig reagents(Supplementary Materials and data not shown) While mAbwith specificity for discrete ferret Ig heavy chain determinantswere unfortunately not generated in this study this exampleserves as motivation for the continued development andcharacterization of ferret Ig-specific reagents

In addition a polyclonal goat anti-ferret Ig reagent withspecificity for IgA IgG and IgM was used to identify ferretB cells in the context of influenza infection using FCM [14]In this study the polyclonal goat anti-ferret Ig (A G andM) failed to label a substantial population of ferret B cellsthat exhibited intracellular staining with the anti-CD79120572mAb (clone HM47) The M120572F-Ig mAb reported here are animprovement over existing reagents and enable the identi-fication of nearly all surface Ig+ ferret B cells (Figure 5(b))and discrimination between Ig120581 and Ig120582-expressing cells(Figure 6(a))

We envision that several of theM120572F-Ig mAb generated inthis study will have applications in basic science and veteri-nary medicine Due to their strong affinity for purified ferretIg and reactivity with denatured heavy (Ig120574) or light chainproteins 4-B10 8-H9 and 11-E3may prove to be most usefulSpecifically these M120572F-Ig mAb should enable the identi-fication of antibody-secreting cells by FCM Additionallythese mAb are likely to have utility as secondary detectionreagents in an array of assays such as ELISA ELISPOT andwestern blot Implementation of these mAb in a variety ofimmunologic assays will also contribute towards the assess-ment of the next-generation of broadly-reactive influenzavaccines in this highly relevant model In summary thegeneration of these M120572F-Ig mAb is an improvement overexisting reagents available to immunologists and opens thedoor to more sophisticated study of ferret B cells

Competing Interests

The authors report no conflict of interests with the resultsreported in this study

Acknowledgments

This work was supported by the Vaccine and Gene TherapyInstitute of Florida and the University of Georgia Addi-tionally The authors thank Lawrence Wysocki for providingprotocols and the SP20 myeloma and Rayleigh Chan andThomas Penrose for their assistance in the production andpurification of reagents used in this work

References

[1] T Enkirch and V von Messling ldquoFerret models of viral patho-genesisrdquo Virology vol 479-480 pp 259ndash270 2015

[2] R Kozak S He A Kroeker et al ldquoFerrets infected with bundi-bugyo virus or ebola virus recapitulate important aspects ofhuman filovirus diseaserdquo Journal of Virology vol 90 no 20 pp9209ndash9223 2016

[3] G A Kirchenbaum D M Carter and T M Ross ldquoSequentialinfection in ferrets with antigenically distinct seasonal H1N1influenza viruses boosts hemagglutinin stalk-specific antibod-iesrdquo Journal of Virology vol 90 no 2 pp 1116ndash1128 2016

[4] M Linster S Van BoheemenMDeGraaf et al ldquoIdentificationcharacterization and natural selection of mutations drivingairborne transmission of AH5N1 virusrdquo Cell vol 157 no 2 pp329ndash339 2014

[5] A Otte A CMarriott C Dreier et al ldquoEvolution of 2009H1N1influenza viruses during the pandemic correlates with increasedviral pathogenicity and transmissibility in the ferret modelrdquoScientific Reports vol 6 Article ID 28583 2016

[6] W Smith C H Andrewes and P P Laidlaw ldquoA virus obtainedfrom influenza patientsrdquo13e Lancet vol 222 no 5732 pp 66ndash68 1933

[7] H Zeng C S Goldsmith T R Maines et al ldquoTropism andinfectivity of influenza virus including highly pathogenic avianH5N1 virus in ferret tracheal differentiated primary epithelialcell culturesrdquo Journal of Virology vol 87 no 5 pp 2597ndash26072013

[8] J A Belser X Lu T R Maines et al ldquoPathogenesis of avianinfluenza (H7) virus infection inmice and ferrets enhanced vir-ulence of eurasianH7N7 viruses isolated fromhumansrdquo Journalof Virology vol 81 no 20 pp 11139ndash11147 2007

[9] L A Zitzow T Rowe T Morken W-J Shieh S Zaki and J MKatz ldquoPathogenesis of avian influenza A (H5N1) viruses in fer-retsrdquo Journal of Virology vol 76 no 9 pp 4420ndash4429 2002

[10] J A Belser A M Eckert T M Tumpey and T R MainesldquoComplexities in ferret influenza virus pathogenesis and trans-mission modelsrdquo Microbiology and Molecular Biology Reviewsvol 80 no 3 pp 733ndash744 2016

[11] NM Bouvier ldquoAnimalmodels for influenza virus transmissionstudies a historical perspectiverdquo Current Opinion in Virologyvol 13 pp 101ndash108 2015

[12] D Y Oh and A C Hurt ldquoUsing the ferret as an animal modelfor investigating influenza antiviral effectivenessrdquo Frontiers inMicrobiology vol 7 article 80 2016

[13] L A Carolan S Rockman K Borg et al ldquoCharacterization ofthe localized immune response in the respiratory tract of ferretsfollowing infection with influenza A and B virusesrdquo Journal ofVirology vol 90 no 6 pp 2838ndash2848 2016

[14] A DiPiazza K Richards F Batarse et al ldquoFlow cytometric andcytokine elispot approaches to characterize the cell-mediatedimmune response in ferrets following influenza virus infectionrdquoJournal of Virology vol 90 no 17 pp 7991ndash8004 2016

[15] A J Leon D Banner L Xu et al ldquoSequencing annotation andcharacterization of the influenza ferret infectomerdquo Journal ofVirology vol 87 no 4 pp 1957ndash1966 2013

[16] N Music A J Reber A S Lipatov et al ldquoInfluenza vaccinationaccelerates recovery of ferrets from lymphopeniardquo PLoS ONEvol 9 no 6 Article ID e100926 2014

[17] T Rowe A J Leon C J Crevar et al ldquoModeling host responsesin ferrets during ACalifornia072009 influenza infectionrdquoVirology vol 401 no 2 pp 257ndash265 2010

[18] K Wang G Wei and D Liu ldquoCD19 a biomarker for B celldevelopment lymphoma diagnosis and therapyrdquo ExperimentalHematology amp Oncology vol 1 no 1 article no 36 2012

[19] J Hombach T Tsubata L Leclercq H Stappert and M RethldquoMolecular components of the B-cell antigen receptor complexof the IgM classrdquo Nature vol 343 no 6260 pp 760ndash762 1990

[20] D Y Mason J Cordell A Tse et al ldquoThe IgM-associated pro-teinmb-1 as amarker of normal andneoplastic B cellsrdquo13e Jour-nal of Immunology vol 147 no 11 pp 2474ndash2482 1991

[21] X Brochet M-P Lefranc and V Giudicelli ldquoIMGTV-QUESTthe highly customized and integrated system for IG and TR


standardized V-J and V-D-J sequence analysisrdquo Nucleic acidsresearch vol 36 pp W503ndashW508 2008

[22] R Nazarpour E Zabihi E Alijanpour Z Abedian H Mehdi-zadeh and F Rahimi ldquoOptimization of Human PeripheralBloodMononuclear Cells (PBMCs) cryopreservationrdquo Interna-tional Journal of Molecular and Cellular Medicine vol 1 no 2pp 88ndash93 2012

[23] T Nakamura H Kubagawa and M D Cooper ldquoHeteroge-neity of immunoglobulin-associated molecules on human Bcells identified by monoclonal antibodiesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 89 no 18 pp 8522ndash8526 1992

[24] W Guo D Smith K Aviszus T Detanico R A Heiser and L JWysocki ldquoSomatic hypermutation as a generator of antinuclearantibodies in a murine model of systemic autoimmunityrdquoJournal of ExperimentalMedicine vol 207 no 10 pp 2225ndash22372010

[25] C-A ReynaudM Descatoire I Dogan F Huetz SWeller andJ-C Weill ldquoIgM memory B cells a mousehuman paradoxrdquoCellular and Molecular Life Sciences vol 69 no 10 pp 1625ndash1634 2012

[26] C J-M Martel and B Aasted ldquoCharacterization of antibodiesagainst ferret immunoglobulins cytokines and CD markersrdquoVeterinary Immunology and Immunopathology vol 132 no 2ndash4pp 109ndash115 2009

[27] J M Brewer M Conacher A Satoskar H Bluethmann andJ Alexander ldquoIn interleukin-4-deficient mice alum not onlygenerates T helper 1 responses equivalent to Freundrsquos com-plete adjuvant but continues to induce T helper 2 cytokineproductionrdquo European Journal of Immunology vol 26 no 9 pp2062ndash2066 1996

[28] B Akerstrom and L Bjorck ldquoProtein L an immunoglobulinlight chain-binding bacterial protein Characterization of bind-ing and physicochemical propertiesrdquo 13e Journal of BiologicalChemistry vol 264 no 33 pp 19740ndash19746 1989

[29] L Bjorck ldquoProtein L A novel bacterial cell wall protein withaffinity for Ig L chainsrdquo Journal of Immunology vol 140 no 4pp 1194ndash1197 1988

[30] C Vettermann and M S Schlissel ldquoAllelic exclusion of immu-noglobulin genes models and mechanismsrdquo ImmunologicalReviews vol 237 no 1 pp 22ndash42 2010

[31] S Bournazos and J V Ravetch ldquoFc120574 receptor pathways duringactive and passive immunizationrdquo Immunological Reviews vol268 no 1 pp 88ndash103 2015

[32] K A Pape J J Taylor R W Maul P J Gearhart and M KJenkins ldquoDifferent B cell populations mediate early and latememory during an endogenous immune responserdquo Science vol331 no 6021 pp 1203ndash1207 2011

[33] C Heilmann and T Barington ldquoDistribution of 120581 and 120582 lightchain isotypes among human blood immunoglobulin-secretingcells after vaccination with pneumococcal polysaccharidesrdquoScandinavian Journal of Immunology vol 29 no 2 pp 159ndash1641989

[34] X Peng J Alfoldi K Gori et al ldquoThe draft genome sequenceof the ferret (Mustela putorius furo) facilitates study of humanrespiratory diseaserdquo Nature Biotechnology vol 32 no 12 pp1250ndash1255 2014

[35] P Sanchez B Nadel and P Cazenave ldquoV120582minusJ120582 rearrangementsare restrictedwithin aVminusJminusCrecombination unit in themouserdquoEuropean Journal of Immunology vol 21 no 4 pp 907ndash911 1991

[36] G Haughton L L Lanier and G F Babcock ldquoThe murinekappa light chain shiftrdquo Nature vol 275 no 5676 pp 154ndash1571978

[37] M-P LeFranc ldquoNomenclature of the human immunoglobulinlambda (IGL) genesrdquo Experimental and Clinical Immunogenet-ics vol 18 no 4 pp 242ndash254 2001

[38] S Barandun A Morell F Skvaril and A Oberdorfer ldquoDefi-ciency of kappa or 120582 type immunoglobulinsrdquo Blood vol 47 no1 pp 79ndash89 1976

[39] C S Ambrose X Wu T Jones and R M Mallory ldquoThe role ofnasal IgA in children vaccinated with live attenuated influenzavaccinerdquo Vaccine vol 30 no 48 pp 6794ndash6801 2012

[40] X Cheng J R Zengel A L Suguitan et al ldquoEvaluation ofthe humoral and cellular immune responses elicited by the liveattenuated and inactivated influenza vaccines and their rolesin heterologous protection in ferretsrdquo Journal of Infectious Dis-eases vol 208 no 4 pp 594ndash602 2013

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


expression [14 16] While CD79120572 is an excellent marker foridentification of B lineage cells because it is an obligate chap-erone for surface expression of B cell receptor (BCR) [19]the epitope recognized by this mAb (clone HM47) requiresintracellular staining and thus does not permit isolation ofviable B cells [20] Alternatively polyclonal antibodies withreactivity against ferret Ig are conducive for surface stainingbut do not exclusively define B cells due to binding of ferretIg by myeloid cells via Fc receptors [14] Moreover neitherapproach enables segregation of ferret B cells on the basis ofheavy chain usage Collectively the suite of available reagentsis still insufficient for an in-depth characterization of thehumoral immune response and specifically the identificationof antibody-secreting cells by FCM To begin to address thisunmet need several novel mAb with specificity for ferretimmunoglobulin (Ig) were generated and characterized todefine their specificity

2 Materials and Methods

21 Animals BALBc mice (female 8ndash10 weeks of age) fromJackson Laboratory (Bar Harbor ME USA) and Fitch ferrets(Mustela putorius furo male or female 6 to 12 months ofage) from Triple F Farms (Sayre PA USA) were housed incage units and fed ad libitum All animals were handled inaccordance with protocols approved by Institutional AnimalCare and Use Committees and were cared for under USDAguidelines for laboratory animals

22 Isolation of Ferret Peripheral Mononuclear Cells Periph-eral blood was collected via cardiac puncture into vacuumcollection tubes containing sodium heparin (Becton Dickin-son Cat 367874) and gently inverted to prevent coagulationBlood was then combined with phosphate buffered saline(PBS) (Corning Cat 21-040-CV) to a final volume of 35mLand overlaid on 10mL Ficoll-Paque PLUS (GE HealthcareCat 17-1440) before centrifugation at 500timesg for 25minwith the brake reduced to its lowest setting using a SorvallLegend XTR (Thermo Scientific Grand Island NY USA)Peripheral blood mononuclear cells (PBMC) at the interfacewere collected washed with PBS and then pelleted (400timesgfor 10min) Following an additional wash with PBS totalcell number and viability was determined by Trypan blueexclusion using theCountess (ThermoFisher Cat C10227)Ferret PBMC were used immediately or resuspended in fetalbovine serum (FBS) (HyClone Cat SH3039603) containing10 DMSO (Thermo Scientific Cat 20688) for long-termstorage Aliquots of 1-2 times 107 viable ferret PBMC were storedin the vapor phase of liquid nitrogen until use and thawing ofcells was according to similarly described methods [22]

23 Purification of Ferret Immunoglobulin Serum from twoferrets (female 7 months of age) was pooled and immu-noglobulin (Ig) precipitated by drop-wise addition of anequal volume of saturated ammonium sulfate solution (41M)(Sigma Cat A4418) while maintaining the solution underconstant agitation at 4∘C Precipitated protein was pelletedby centrifugation at 115000 rpm for 20min at 4∘C and thendissolved in PBS The protein solution was then transferred

into a Slide-A-Lyzer dialysis cassette (ThermoFisher Cat66030) and dialyzed against PBS at 4∘C for three days withdaily buffer exchanges Subsequently the protein solutionwasclarified by centrifugation at 6000 rpm for 10min at 4∘C andthen passed through a 02 120583m syringe filter (ThermoFisherCat 09-719C) This material is referred to as crude ferretIg Ferret Ig was further purified by affinity chromatographyusing ProteinAG (ThermoFisher Cat 20423) Briefly crudeferret Ig protein solution was applied to Protein AG and thecolumn (Bio-Rad Cat 7311550) was washed by gravity flowwith PBS Fractions (2mL) were collected and absorbance(280 nm) was monitored using a PowerWave XS spectrom-eter (Biotek Winooski VT USA) Once wash fractionsreturned to baseline ferret Ig protein was eluted by additionof 01Mglycine pH 25 (Amresco Cat M103) Eluted proteinwas immediately neutralized with 200120583l 15M Tris pH88 (Amresco Cat M151) and protein containing elutionfractions were pooled buffer exchanged into PBS containing005 sodium azide (Sigma Cat S2002) and concentratedusing a Spin-XUF filter (Sigma Cat CLS431489)Thismate-rial is referred to as purified ferret Ig Protein concentrationsof the crude and purified ferret Ig solutions were determinedaccording to the manufacturersquos instructions using a microBCA assay kit (ThermoFisher Cat 23235)

24 ProteinGel Electrophoresis To assess purity of the respec-tive ferret Ig containing protein solutions 5120583g of crude orpurified ferret Ig was loaded into Bolt 10 Bis-Tris Plusprecast protein gels (ThermoFisher Cat NW00102) andresolved at 150V for 50min Protein samples were dilutedin either 2x Laemmli sample buffer (Bio-Rad Cat 161-0737)with or without 120573-mercaptoethanol (JT Baker Cat P62405)or 6x SDS-sample buffer (reducing) (Boston BioProductsCat BP-111R) Reduced samples were heated at 100∘C for10min and placed on ice prior to loading Gels were stainedwith PageBlue protein staining solution (ThermoFisherCat 24620) and then destained in water before imagingusing the myECL Imager (ThermoFisher Waltham MAUSA) Spectra Multicolor Broad Range Protein Ladder(ThermoFisher Cat 26634) was included in all gels and usedfor molecular marker reference

25 Generation of Murine Monoclonal Antibodies FemaleBALBc mice were immunized with 100 120583g of purified ferretIg containing the Imject alum adjuvant (Thermo ScientificCat 77161) via the intraperitoneal route Serumwas collectedvia the submandibular vein on day 21 and assessed by ELISAfor antibody reactivity (refer to ELISA subsection) The twomicewith the highest antibody titer received a booster immu-nization via the intraperitoneal route containing 100120583gof purified ferret Ig in PBS on Day 28 Three or fourdays after the booster immunization splenocytes wereharvested and used to perform a fusion with the SP2Omyeloma (kindly provided by Dr Lawrence Wysocki Uni-versity of Colorado Denver) using polyethylene glycol 1450(ATCC Cat 50-X) Hybridomas were selected by addi-tion of hypoxanthine (Acros Organics Cat 1220100) andazaserine (Sigma Cat A4142) at a final concentration of200120583M and 115 120583M respectively in RPMI 1640 (Sigma
















3 Results




140 kDa260 kDa

Lane

2

Lane

3

Lane

4

Lane

5

Lane

1100 kDa

70 kDa

50 kDa

40 kDa

35 kDa

25 kDa

15 kDa

10 kDa

Ig휇

Ig훾

Ig휅휆






Ferret Ig

101 102 103 104100


4-E36-H5

6-B56-B78-H9

11-E3

0

1

2

3

4

OD414








Isotypecontrol

50 kDa

30 kDa25 kDa

Lane

1La

ne 2

Lane

3La

ne 4

Lane

5

(a)mAb 4-B10

(b)mAb 8-H9

(c)mAb 11-E3

(d)








G훼M-IgG onlyG

oat a

nti-m

ouse

IgG

Ale

xa F

luor

647


0

102

103

104

105

0 103 104 105

(a)


Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(b)

mAb 4-B10

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)


mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)




mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































3 Results




140 kDa260 kDa

Lane

2

Lane

3

Lane

4

Lane

5

Lane

1100 kDa

70 kDa

50 kDa

40 kDa

35 kDa

25 kDa

15 kDa

10 kDa

Ig휇

Ig훾

Ig휅휆






Ferret Ig

101 102 103 104100


4-E36-H5

6-B56-B78-H9

11-E3

0

1

2

3

4

OD414








Isotypecontrol

50 kDa

30 kDa25 kDa

Lane

1La

ne 2

Lane

3La

ne 4

Lane

5

(a)mAb 4-B10

(b)mAb 8-H9

(c)mAb 11-E3

(d)








G훼M-IgG onlyG

oat a

nti-m

ouse

IgG

Ale

xa F

luor

647


0

102

103

104

105

0 103 104 105

(a)


Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(b)

mAb 4-B10

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)


mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)




mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















140 kDa260 kDa

Lane

2

Lane

3

Lane

4

Lane

5

Lane

1100 kDa

70 kDa

50 kDa

40 kDa

35 kDa

25 kDa

15 kDa

10 kDa

Ig휇

Ig훾

Ig휅휆






Ferret Ig

101 102 103 104100


4-E36-H5

6-B56-B78-H9

11-E3

0

1

2

3

4

OD414








Isotypecontrol

50 kDa

30 kDa25 kDa

Lane

1La

ne 2

Lane

3La

ne 4

Lane

5

(a)mAb 4-B10

(b)mAb 8-H9

(c)mAb 11-E3

(d)








G훼M-IgG onlyG

oat a

nti-m

ouse

IgG

Ale

xa F

luor

647


0

102

103

104

105

0 103 104 105

(a)


Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(b)

mAb 4-B10

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)


mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)




mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Isotypecontrol

50 kDa

30 kDa25 kDa

Lane

1La

ne 2

Lane

3La

ne 4

Lane

5

(a)mAb 4-B10

(b)mAb 8-H9

(c)mAb 11-E3

(d)








G훼M-IgG onlyG

oat a

nti-m

ouse

IgG

Ale

xa F

luor

647


0

102

103

104

105

0 103 104 105

(a)


Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(b)

mAb 4-B10

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)


mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)




mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















G훼M-IgG onlyG

oat a

nti-m

ouse

IgG

Ale

xa F

luor

647


0

102

103

104

105

0 103 104 105

(a)


Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(b)

mAb 4-B10

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(c)

mAb 8-H9

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(d)

mAb 6-B7

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47


0

102

103

104

105

0 103 104 105

(e)

mAb 11-E3

Goa

t ant

i-mou

se Ig

GA

lexa

Flu

or 6

47

0

102

103

104

105


0 103 104 105

(f)


mAb

6-B

5 BI

O +

SA-

PE

CD79훽 PerCP-Cy55

minus102

102

103

104

105

0

103 104 1050

(a)

CD79훽 PerCP-Cy55

mAb

6-B

7 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)

CD79훽 PerCP-Cy55

mAb

11-

E3 B

IO +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(c)




mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















mAb

8-H

9 (D

yLig

ht 6

50)

minus102

102

103

104

105

0

103 104 1050

(a)


mAb

4-E

3 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(b)


mAb

4-D

11 B

IO +

SA-

PE

103 104 1050

minus102

102

103

104

105

0

(c)


mAb

6-H

5 BI

O +

SA-

PE

minus102

102

103

104

105

0

103 104 1050

(d)






4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















4-B10 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

of m

ax si

gnal


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(a)

4-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(b)

8-H9 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(c)

11-E3 (5 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

010

000

2000

0

0

25

50

75

100

125

o

f max

sign

al


6-B5 (IgH)6-B7 (IgH)11-E3 (IgH)

(d)

6-B7 (100 ngwell)


98

195 39

78

156

312

5625

1250

2500

500

0625

012

500

2500

0500

00

0

25

50

75

100

125

o

f max

sign

al



(e)



4 Discussion






Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Prot

ein

L D

yLig

ht 6

50


mAb

8-H

9 BI

O +

SA-


Ig휅(4-B10)

Ig휆(8-H9)

0

20

40

60

80

100

o

f B ce

lls

minus1020

102

103

104

105

minus1020

102

103

104

105

0 103 104 105 0 103 104 105

239 plusmn 217

172 plusmn 234

824 plusmn 24

lowastlowastlowast




























Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































Competing Interests


Acknowledgments


References















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















generation of monoclonal antibodies against immunoglobulin...

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