research article characterization of human colorectal...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 716289 12 pageshttpdxdoiorg1011552013716289

Research ArticleCharacterization of Human Colorectal CancerMDR1P-gp Fab Antibody

Xuemei Zhang12 Gary Guishan Xiao3 and Ying Gao2

1 The Medical College of Dalian University Dalian Economic amp Technical Development Zone Dalian 116622 China2Department of Biochemistry and Molecular Biology Dalian Medical University 9 Western Section Lvshun South StreetLvshunkou District Dalian 116044 China

3Departments of Medicine and Medical Microbiology Creighton University 601 N 30th Street Omaha NE 68131 USA

Correspondence should be addressed to Ying Gao gaoying822hotmailcom

Received 22 May 2013 Accepted 1 September 2013

Academic Editors Y Nishita T Robak and Y Xi

Copyright copy 2013 Xuemei Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In this study the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mousemonoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp

21by phage display

technology Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21with five rounds of panning A number of clones expressing Fab bound to P-gp

21 showing neutralized activity in vitro were

isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21and human colorectal

cancer tissue homogenate resulting in identification of an optimal recombinant Fab clone (Number 29) Further characterizationby recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29by Isopropyl 120573-D-1-thiogalactopyranoside (IPTG) After purified by HiTrap Protein L the specificity of the Fab antibody to P-gp21

was also confirmed Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope(ALKDKKELEGSGKIAT) comprising residues 883ndash898 within the transmembrane (TM) domain of human P-gp but also thebinding ability with it was verified The clinical implication of our results for development of personalized therapy of colorectalcancer will be further studied

1 Introduction

Failure of chemotherapy resulting from a multidrug resis-tance (MDR) remains the major obstacle during cancertreatment Development of effective screening tools for per-sonalized therapy of cancer will enhance the response rate ofthe patients to the designated therapeutic regimen One ofthe gene products involved in MDR is P-glycoprotein (P-gp)P-gp is a 170 kD plasma membrane efflux pump protein andbelongs to the ATP-binding cassette superfamily of transportproteins [1] Comparative sequence analysis of P-gp withother ABC family members shows that it consists of twotransmembrane (TM) domains each containing six putativeTM segments followed by an ATP-binding consensus motifand two cytoplasmic nucleotide-binding domains (NBDs)The TMs of P-gp are found mainly responsible for bindingto and transporting a broad spectrumof drugs Photolabeling

andmutational studies indicate that the drug binding domainis within the TM domains of P-gp and drug transport ispowered by hydrolysis of ATP at the two cytoplasmic NBDs[1 2] Structure-function analyses of P-gp suggested that twosubunits of P-glycoprotein are involved in the selection of itsdrug substrate andor specificity of MDRmodulator Crucialamino acid residues of P-gp are localized within or near theTM region [3 4] P-gp functions as an efflux pump expellingdrugs out of tumor cells resulting in a decrement intracellularconcentration of cytotoxic drugs Overexpression of P-gp isassociated with poor clinical outcome [5ndash7] Identificationof the tumor cells sensitive to antineoplastic drugs may helpfor the selection of the antineoplastic agents [8] Althoughmethyl thiazolyl tetrazolium (MTT) can provide direct obser-vation on the tumor cells sensitive to antitumor drugs itis a time-consuming process Methods used extensively fordetection of the P-gp protein expression such as ELISA

2 The Scientific World Journal

immunohistochemical method (IHCA) and flow cytometry(FCM) depend on the reaction of the P-gp protein to itscorresponding monoclonal antibody Nevertheless use ofthe intact monoclonal antibodies may provide false positivesignals in detection of the tumor cells since the Fc fragmentsof IgG may bind to the Fc receptors on the surface of normalcells as well Thus use of the Fab antibody may be superiorto reducing the false signal resulting from the intact antibody[9 10] Meanwhile Fab is smaller than IgG in size facilitatingtrafficking of the antigen across cell membrane and mayprovidemore information than the intact antibody during theICHA process [11 12]

In this communication we constructed and characterizedthe phage-displayed mouse Fab against human MDR1P-gp

2 Materials and Methods

21 Animals The study was conducted in accordance withnational guidelines for care and use of animals All micewere purchased by the DalianMedical University LaboratoryAnimal Center (approval number LA 2009-008)The animalsused in the study were age-matched mature BALBc femalemouse weighing approximately 18ndash22 g

22 Construction of Phage-Displayed Mouse Fab AntibodyLibrary against Human Colorectal Cancer MDR1P-gp SixBALBC female mice were injected subcutaneously (02mL10 g) with 05mgmL human colorectal cancer homogenateemulsified in complete Freundrsquos adjuvant (CFA) The samecolorectal cancer homogenate and aliquot of Freundrsquos incom-plete adjuvant were subcutaneously injected on the seventhand fourteenth days respectively Seven days after the lastimmune blood samples were collected and used for deter-mination of the antibody titer by ELISA When the antibodytiter reached above 1 512 the spleens of the mice wereremoved immediately frozen by liquid nitrogen and usedfor extraction of total RNA Specific cDNA was synthesizedfrom RNA samples with random primer using the RevertAidFirst Strand cDNA Synthesis Kit (Fermentas) according tothemanufacturerrsquos instructionsThe Fd chain Lambda chainand Kappa chain were then synthesized from the cDNAwith 37 pairs of primers which were designed to annealwith mouse FR1 and CH

1or CL regions with high fidelity

Taq DNA polymerase (Fermentas USA) respectively PCRamplification was performed for 35 cycles (1min at 94∘C1min at 55∘C and 1min at 72∘C) in a Thermal Cycler S100(Bio-Rad CA USA)

PCR products were pooled into independent collectionsof Fd chain 120581 chain and 120582 light chain and purified by usingan AxyPre DNA Gel Extraction Kit (Axygen CA USA)The recombinants with Fd were performed according tothe method described as ldquoAntibody Phage Display Methodsand Protocolsrdquo [13] The recombinants with light chain andpGEM-T were firstly digested by Xba I and Sac I (Fermen-tas USA) then ligated into the Fd-pComb3 library andtransfected into E coli XL1-Blue cells (Stratagene CA USA)repeatedly to obtain the Fab library (gt106 CFU)The presenceand size of the inserts were further confirmed sequentiallywith Xho ISpe I and Xba ISac I

23 Cloning and Expression of the P-gp TransmembraneRegion A gene encoding 185 amino acids covering P-gptransmembrane region was isolated from the colon can-cer tissues and amplified by PCR using the sense primer51015840 CCGGAATTCCTCACCAAGCGGCTCCGAT 31015840 andthe antisense primer 51015840 CCGCTCGAGGAGTTTATGTGC-CACCAAGTAG 31015840 and then cloned into a pET28a (+)vector The recombinant was further verified by sequencingThe positive recombinants were selected and amplified byadding 10mM IPTG for 6 h at 30∘C Cell pellets werecollected by centrifugation at 14006timesg for 3min Proteinwasextracted from the cell pellets by using the nickel column (GEHealthcare CA USA) The purified P-gp

21was verified by

Western blot with the His-tag monoclonal antibody (1 2000)(Protein Tech CA USA)

24 Phage Display and Screening of the Clones Phagemid res-cuing and the Fab displayed library screeningwere conductedaccording to the protocol described [14] Phage particlesexposing antibody fragments were rescued from the Fablibrary with helper phage with VCSM13 helper phage (26 times1011 pfumL) on a 20mL scale The resulting phage mixtureswere used for binders selection on Nunc-Immuno MaxiSorpflat-bottomedmicrotiter plates (NuncDenmark) coatedwith49 120583g100 120583L of P-gp

21dissolved in bicarbonate buffer (pH

96) per well overnight at 4∘C Coated plates were incubatedin 3 BSA in PBS 300 120583L per well for 2 hr at 37∘C forblocking Then 200120583L Fab phage particles prepared abovewere incubated in microtiter plates for 1 h at RT Nonboundphages were washed away for 1 5 10 15 to 20 times perround with PBS containing 01 Tween 20The bound phageparticles were then eluted with 200120583L of elution buffer (01MHCl-glycine pH 22) per well and immediately neutralizedwith 14 120583L of 2M Tris base solution Collected phages wereused to infect E coliXL1-Blue cells in an exponential state andamplified as described above Rescued phage particles wereused to start a new selection round in the same conditionsaccording to the same protocol as described above [14]The phagemid titer was checked by counting the colony-forming unit (CFU) of the phagemid infected by XL1-Bluecells just before and after each panning After the fifth roundof screening a total of 10 colonies were picked randomly andverified by a double digestion using enzymes Xba ISac I andXho ISpe I respectively

25 Further Verification of the Anti-P-gp Positive Clones byELISA The positive Fab colonies were picked to test theirP-gp21

recognition properties Afterwards aliquots (20120583L)of the positive clones were transferred to 10mL of LB-Amp-Tet and further cultured until their A600 reached the sameas 04 Soon after IPTG was added to a final concentrationof 005mM and kept shaking at 220 rpm at 28∘C for sixhours The supernatants of each clone after centrifugation at14006timesg for 2min at 4∘Cpellets were collected and sonicatedfor 10min for ELISA

Moiety of the purified P-gp21

(1120583g120583L) and human col-orectal cancer homogenate (2120583g120583L) was incubated in 50120583Lbicarbonate buffer (pH 96) at 4∘C overnight respectively

The Scientific World Journal 3

After blocking with 3 BSA for 2 hrs at 37∘C an equal ofthe soluble protein from each clone prepared above wasmixed well for 1 hr at 37∘C After washing with 01 Tween20 in PBS alkaline-phosphatase- (ALP-) labeled horse anti-mouse IgG antibody (1 2000) (Vector Laboratories IncCA USA) was added as the secondary antibody and p-nitrophenyl phosphate (PNPP) as a chromogenic agent wascoincubated with each blot The clone with pComb3 wasserved as a negative control meanwhile the E coli XL1-Bluewas served as a black control The absorbance was measuredat 405 nm with a microplate reader (Model 550 Bio-RadCA USA) Meanwhile the optimal clone was further verifiedby the analysis of Western blot Mouse anti-His antibody(1 2000) was used as a positive control and crude cell extractof pComb3 clone was served as a negative control Horseanti-mouse antibodies conjugated to alkaline phosphatase asthe secondary antibody (1 2000) and BCIPNBT (AmrescoCA USA) as a chromogenic agent Data obtained from theWestern blot were analyzed by Bio-Rad Quantity One 1DAnalysis software version 11 (Bio-Rad CA USA) PlasmidDNA from the optimal clone was purified and the insertswere completely sequenced and the deduced amino acidsequences were compared with DNA databank data usingthe BLAST program (National Center for BiotechnologyInformation USA) to ascertain its sources

26 Production of Soluble Fab Fragments The recombinantplasmid DNA from the clone number 29 was digested withSpe I and Nhe I (MBI Fermentas USA) for 2 h at 37∘C toremove the gIII fragment from pComb3 purified by using gelelectrophoresis and then self-ligated to build constructs forexpression of soluble recombinant FabAfter the recombinantwas identified by Not I digestion the clone was suspendedin LB medium containing 100 120583gmL of ampicillin andincubated overnight at 30∘C Afterward 4mL of this culturewas inoculated in 400mL of LB medium with ampicillinat 100 120583gmL and induced by isopropylthio-b-galactoside atthe final concentration of 05mM until the culture reachedexponential growth and then further incubated for 8 h at28∘C The E coli cells were harvested by centrifugation at2218timesg for 15min at 4∘C and the pellet was suspended with20mL of PBS and sonicated on ice Crude cell extract withFab fragments was obtained by centrifugation at 8873timesg for30min at 4∘C

27 Purification of Fab The supernatant containing Fabprepared above was filtered by 022mm filter membraneThe filtered solution was loaded onto Capto-L agarose chro-matography column (HiTrap Protein L GE) with the flowvelocity of 1mLmin After washing out the unbound proteinthe Fab was eluted out with sodium acetate buffer (pH23) and neutralized with Tris-HCl (pH 80) immediatelyBoth flow-through unbound protein and eluted protein werecollected for further verification by SDS-PAGE

28 Analysis of the Characters of the Anti-P-gp Fab FragmentExpressed in E coli XL1-Blue After purification Fab concen-tration was determined using a BCA protein assay kit (Pierce

Biotechnology USA)The specificity of the purified Fab to P-gp21was also analyzed byWestern blot using goat anti-mouse

IgG conjugated to HRP (1 3000) (Southern Biotech CAUSA) The moiety of BSA and the 15 kDa peptide expressedby BL

21(prepared by our lab) were served as the negative

controlP-gp21harboring three epitopeswas chosen as the antigen

according to its antigenicity estimated by using BepiPred10b Server (Figure 1)Three peptides with strong antigenicitycoupled to bovine serum albumin were synthesized (ChinaPeptides Co Ltd Shanghai China) for selection of the Fab A96-well microtiter plate (Nunc Denmark) was coated withaliquot of BSA 10-peptide-BSA 12-peptide-BSA and 16-peptide-BSA at 1 120583g120583L and then incubated at 4∘CovernightAfterwashing away unbound antigen the platewas incubatedin the blocking solution (3 BSA in PBS) at 37∘C for 1 hThe plate was then incubated with the Fab antibody preparedabove (approximately 6 120583gmL) at 37∘C for 1 h After ten-timewashwithTBS the platewas then incubatedwith fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgG(Shanghai Meilian Biological Technology Co Ltd China)(1 1000) as secondary antibody for 1 hour at 37∘C Crude cellextract of pComb3 cell was used as a negative control andthe secondary antibodies were used only as a backgroundcontrol in the detection system The relative fluorescenceunit (RFU) was measured by a Multimode Reader (LB941Berthold Technologies Germany) with excitation spectrumat 490 nm and emission spectrum at 520 nm

The specificity of the Fab antibody was measured by asequential dilution of the Fab fragments immobilizedwith 16-peptide-BSA and BSA microplates as mentioned above

3 Results

31 Quantitative Analysis of Total RNA Total RNA extractedfrom an immunized mouse spleen was measured quantita-tivelyThe quality of the RNA samples was estimated by usingthe ratio of A260A280 and agarose gel electrophoresis Theratio of A260A280 for all RNA samples was in the range of18ndash22 suggesting that a highly qualified RNA sample wasobtained Quality of total RNA samples was further verifiedby 1 agarose gel electrophoresis showing clear bands in 28SRNA and 18 S RNA (Figure 1(a))

32 Construction of Fd Library To construct IgG heavy chainrecombinant Fd-pComb3 we first amplified IgG light chainsand Fds by PCR Figures 1(b) and 1(c) showed that IgGlight chains and Fds were successfully amplified with anapproximate size of 650 bp and 730 bp separately as shownin 1 agarose gel Then light chains and Fd fragmentswere purified for the next library construction Fd-pComb3recombinant was constructed followed by purification andligation The library of Fd-pComb3s was transformed intoTop 10 competent bacteria by heat shock

33 Construction of Fab Library The recombinants were firstconstructed by connection of the light chains with T carriersand then transformed into TOP 10 cells and cultured at 37∘C


5000

2000

1000

(bp) M 1

28S

18S

(a)

(bp) M 1 1918171615141312111098765432 20

750500

(b)

(bp) M 1 1615141312111098765432 17

750500

(c)

Figure 1 Agarose gel electrophoresis of total RNA and the PCR products of IgG light chains (a) M RL6000 RNAmarker lane 1 total RNAof mouse spleen (b) PCR products of the 120581 chains (lanes 1sim18) PCR products of 120582 chains (lanes 19sim20) (c) PCR products of the Fd chains(lanes 1sim17)

overnight During the blue-white selection when the ratio ofwhite clonesblue clones exceeded 95 they were collectedand reached 18 times 105 CFU after several transductions Afterdigestion of Fd-pComb3 and light chain-T recombinantswith Xba ISac I the recombinants of Fab-pComb3 weretransformed into E coli XL1-Blue successfully and the finalcontent of the Fab library (gt106 cfu) was achieved Twentyclones were picked from the Fab library randomly anddigested by Xba ISac I and Xho ISpe I simultaneouslyto verify the insert ratio of Fab (Figures 2(a) and 2(b))The content of mouse Fab antibody library against humancolorectal cancer P-gp was reached by 247 times 106 cfu Theinsert ratio of the light chain the Fd chain and the Fab chainwas reached by 90 80 and 72 respectively

34 Cloning of Human Multidrug Resistant Protein P-gp21

To clone the MDR P-gp21 the P-gp transmembrane region

(amino acid 784sim968) adjoining the cytoplasmic nucleotide-binding domain (NBD) was chosen as the target accordingto its antigenicity predicted by using BepiPred 10b Server(Figure 3) After total RNA was extracted from human col-orectal cancer the target gene segments were then amplifiedsuccessfully by PCR (Figures 4(a) and 4(b)) The target gene

and pET28a (+) vector were digested by EcoR I and Xho Iand collected by gel electrophoresis (Figure 4(c)) The targetgene was cloned into pET28a (+) vector and transformedinto BL21 (DE3) cell The single clone was randomly pickedand verified by EcoR I and Xho I digestion (Figure 4(d))The sequence of the gene and its encoded amino acid of thepositive clone were confirmed and compared using NCBIdatabase Although there are two nucleotides different fromthe retrieval sequence and no termination codon existedits translated amino acid has no effect on its antigenicity(Table 1)

We optimized the production of P-gp21

and found thatits expression was achieved maximally at 10mM IPTGunder 30∘C for 6 hours (Figure 5(a)) The purified P-gp

21

protein from the cultured bacteria was prepared at a finalconcentration of 52120583g120583L used for subsequent panning(Figure 5(b)) Meanwhile the purified P-gp

21protein was

verified by Western blot analysis as shown in Figure 5(c)

35 Biopanning and Identification of Fab Phage AntibodyLibrary After infection of the Fab antibody library by thehelper phage VCSM13 the phage-displayed Fab antibodylibrary was obtained and the content was reached by 264 times109 pfu120583LThe Fab phage antibody library was then screened


(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(a)

(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(b)

(bp) M 1 1098765432 M 1998400 2998400 3998400 4998400 5998400 6998400 7998400 8998400 9998400

750500

10998400

(c)

Figure 2 Verification of the Fab clones before and after panning by double-enzyme digestion (a) Before panning Fab clones (number1simnumber 10) were digested by Xba ISac I (lanes 1sim10) and by Xho ISpe I (lanes 11015840sim101015840) (b) before panning Fab clones (number 11simnumber20) were digested by Xba ISac I (lanes 1sim10) and by Xho ISpe I (lanes 11015840sim101015840) (c) after panning Fab clones (number 1simnumber 10) weredigested by Xba ISac I (lanes 1sim10) and Xho ISpe I (lanes 11015840sim101015840)

Table 1 BLAST of the P-gp21 sequence with that retrieved from NCBI

Nucleotide sequence of P-gp21

CTCACCAAGCGGCTCCGATACATGGTTTTCCGATCCATGCTCAGACAGGATGTGAGTTGGTTTGATGACCCTAAAAACACCACTGGAGCATTGACTACCAGGCTCGCCAATGATGCTGCTCAAGTTAAAGGGGCTATAGGTTCCAGGCTTGCTGTAATTACCCAGAATATAGCAAATCTTGGGACAGGAATAATTATATCCTTCATCTATGGTTGGCAACTAACACTGTTACTCTTAGCAATTGTACCCATCATTGCAATAGCAGGAGTTGTTGAAATGAAAATGTTGTCTGGACAAGCACTGAAAGATAAGAAAGAACTAGAAGGTTCTGGGAAGATCGCTACTGAAGCAATAGAAAACTTCCGAACCGTTGTTTCTTTGACTCAGGAGCAGAAGTTTGAACATATGTATGCTCAGAGTTTGCAGGTACCATACAGAAACGCTTTGAGGAAAGCACACATCTTTGGAATTACATTTTCCTTCGCCCAGGCAATGATGTATTTTTCCTATGCTGGATGTTTCCGGTTTGGAGCCTACTTGGTGGCACATAAACTC

Amino acid sequence ofP-gp21

LTKRLRYMVFRSMLRQDVSWFDDPKNTTGALTTRLANDAAQVKGAIGSRLAVITQNIANLGTGIIISFIYGWQLTLLLLAIVPIIAIAGVVEMKMLSGQALKDKKELEGSGKIATEAIENFRTVVSLTQEQKFEHMYAQSLQVPYRNALRKAHIFGITFSFAQAMMYFSYAGCFRFGAYLVAHKL

The sequences with underlines are three epitopes analyzed by BepiPred 10b Server the nucleotides located as number442 and number 484 (italics) were different from the retrieval sequences and their translated amino acid changed fromserine to alanine as a result The alanine located as number 148 and number 162 has no effect on the antigenicity of thethree epitopes

by five rounds with the purified P-gp21

protein as shownin Table 2 The library was enriched by sequential panningusing the P-gp

21immobilized onmicroplatesThe number of

colonieswas calculated inCFU in each panning round beforeand after selection by antigen and the library enrichmentfactor was then estimated (Table 2) An enrichment factor of34-fold was achieved in the second panning and only 012092 and 086 in the third the forth and the fifth panningthus indicating that phage maximal production occurred inthe second panning round Ten phage clones were pickedrandomly after 5-round panning and each plasmid DNAwas extracted and digested by Xba ISac I and Xho ISpe I

(Figure 2(c)) respectively The results show that comparedwith the primary Fab library five-round panning made theinsert ratio of the Fab chain increase from 72 to 100

36 Identification of the Optimal Clone Expressing Fab againstP-gp21 To identify the ideal clone expressing Fab against

P-gp21 these positive clones were then amplified up to the

same OD600

the expressed protein in the supernatant of eachclone was further verified by ELISA The expression levelof the Fab in the supernatant from the cultured clones wasexamined when either the P-gp

21or the human colorectal

cancer homogenate was used as an antigen The results of


Table 2 The panning results of Fab phage antibody library

Panning round Phagemid input (CFUamL) Phagemid output (CFUbmL) bound (10minus2)c Enrichmentd

1 13 times 1013 146 times 109 1102 103 times 1012 390 times 109 3790 34363 141 times 1011 638 times 107 452 0124 106 times 1011 440 times 107 415 0925 658 times 1010 770 times 106 117 086aNumber of CFU (colony-forming units) of phagemids incubated with 16-peptide-BSAbTotal number of CFUs of phage contained in elutesc(Phage outputphage input)100dFold increase in bound compared to the previous round of panning

Figure 3The epitopes analysis of 784sim968 amino acids of P-gp Blueunderlines amino acids the possible epitope of target protein

the two ELISA tests showed identically that clone number19 and number 29 were significantly higher than the control(Figure 6) However only clone number 29 was furtheridentified by Western blot analysis as an optimal clone whenit was used as primary antibody (Figures 7(a) and 7(b))Number 29 was sequenced for both light and heavy chainsand aligned by a nucleotide blast program to confirm thatthe sequences with the higher identity weremouse IgG kappachain and mouse immunoglobulin subtype of IgG2a whichare presented in Tables 3 and 4 respectively

37 Production and Purification of the Soluble Fab FragmentsThe recombinant plasmid DNA samples of the clone number29 were extracted and digested with Spe I and Nhe I toremove gIII (680 bp) which belongs to the bacteriophage(Figure 8(a)) The rescued fragment with 4700 bp was self-ligated and transformed into XL1-Blue cells After the recom-binant was identified by Not I digestion the positive cloneexpressing Fab antibody was induced by IPTG (Figure 8(b))Overexpression of the Fab antibody was achieved success-fully under the optimized condition (Figure 9(a)) The Fabantibody from the crude cell extract was purified by affinitychromatography with recombinant protein L ligand owing tostrong affinity for the variable region of an antibodyrsquos kappalight chain showing that concentration of the Fab antibodyat 150mgL was reached (Figure 9(b))

Table 3 Light chain homology analysis of displaying number 29clone the three sequences with higher homology (GenBank)

Nucleotide blastQuery length 635Query ID lcl|10881 Mus musculus immunoglobulin kappa chainconstant region mRNAa

IgBLASTIdentidade (total) CDR1-IMGT CDR2-IMGT CDR3-IMGT968 (274283) 944 (1718) 100 (99) 895 (1719)aGenBank BC0917541

Table 4 Fd homology analysis of clone number 29 displaying thesequences with higher homology (GenBank)

Nucleotide blastQuery length 371 (CH1) query id lcl|31099 Mus musculusPAC-Fab mRNA for immunoglobulin gamma heavy chainvariable region partial cdsb

Query length 696 query id lcl|62943 M musculus mRNA formonoclonal antibody heavy chain gamma 2ac

IgBLASTIdentidade (total) CDR1-IMGT CDR2-IMGT CDR3-IMGT959 (282294) 917 (2224) 833 (2024) 100 (66)bGenBank X704231cGenBank AB6787241

38 Characterization of the Anti-P-gp Fab Fragment Expressedin E coli XL1-Blue To further confirm the purified Fab P-gp21

was detected and estimated to be 21 kDa in size whenneither BSA nor 15-kDa peptide was used as the antigen byWestern blot analysis (Figure 7(b))

Three peptides with strong antigenicity which belong toP-gp21

coupled to bovine serum albumin were synthesizedand used for identifying the exact epitope recognized by theFab using an indirect immunofluorescence assay Comparedto either BSA 10-peptide-BSA or 12-peptide-BSA the Fabshowed high specificity bound to 16-peptide-BSA (Figure 10)These results were further confirmed by indirect immunoflu-orescence assay as shown in Figure 11

An aliquot of BSA 10-peptide-BSA 12-peptide-BSAand 16-peptide-BSA at 1 120583g120583L was coated on a 96-wellmicrotiter plate The anti-P-gp Fab (approximately 6 120583gmL)


(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)

Figure 4 Construction and verification of the P-gp recombinant (a) The total RNA of human colorectal cancer M DL2000 DNA markerlane 1 total RNA of human colorectal cancer (b) PCR amplification of P-gp

21gene M DL2000 DNA Marker lane 1 blank control lane

2 target gene of P-gp21 (c) pET28a (+) and P-gp

21gene digested by EcoR IXho I double enzyme M DL10000 DNA Marker lane 1 the

digested pET28a (+) lane 2 the digested P-gp21target gene (d) Positive clone of P-gp

21verified by double-enzyme cleavage M DL10000

DNAMarker lane 1 positive clone of P-gp21

72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)

Figure 5 Expression purification and verification of the P-gp21 (a) SDS-PAGE image of the P-gp

21expression induced by IPTG before

induction (lane 1) induced for 1 hour (lane 2) induced for 2 hours (lane 3) induced for 3 hour (lane 4) and induced for 4 hours (lane 5) (b)purification of the P-gp

21protein by HiTrap HP before passing Ni affinity column (lane 1) sample after passing Ni affinity column (lane 2)

the washout of the Ni affinity column with 50mM imidazole (lane 3) the washout of the Ni affinity column with 150mM imidazole (lane 4)and the washout of the Ni affinity column with 300mM imidazole (lane 5) (c) P-gp

21protein verified byWestern blot the recombinant with

P-gp21before induction (lane 1) purified P-gp

21protein (lane 2) antigen (P-gp

21protein) primary antibody (mouse anti-His antibody) and

secondary antibody (HRP-labeled goat anti-mouse IgG antibody)

was added and incubated at 37∘C for 1 h Fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgGdiluted 1 1000 is a secondary antibody for signal detectionand crude cell extract of pComb3 cell was used as a negativecontrol and secondary antibodies only as a backgroundcontrol The relative fluorescence unit (RFU) was measuredwith an exitation spectrum at 490 nm and emission spectrumat 520 nm

Data for anti-P-gp Fab and cell extract of pComb3cell were subjected to ANOVA differences between meanswere determined using the least significant difference (LSD)statistic (119875 lt 005)

Microplates were coated with 16-peptide-BSA and BSAat 1120583gmL respectively Fab was titrated down by twofold

dilution starting at 6120583gmL of antibody concentration Thesecond antibody was fluorescein-isothiocyanate- (FITC-)conjugated goat anti-mouse IgG RFU was measured withan exitation spectrum at 490 nm and emission spectrum at520 nm

4 Discussion

Intrinsic or acquired resistance to a broad spectrum ofchemotherapeutic agents often occurred in cancer patientswho receive chemotherapy resulting in failure of chemother-apy The rate of tumor response to drug depends on anumber of factors including pharmacokinetic factors tumortype and biology host response drug sanctuaries and drug


001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm

Diagram of ELISA test coated by P-gp21 protein

(a)

0010203040506070809

OD405nm

Diagram of ELISA test coated by colorectal cancer homogenate

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)

Figure 6The optimal clone was checked by ELISA (a) Diagram of ELISA (plate wells were coated by P-gp21protein) (b) diagram of ELISA

(plate wells were coated by colorectal cancer homogenate)The 119909-axis shows the clone number the 119910-axis shows the ratio of the ELISA signal(A450) in the presence of 1 120583g120583L purified P-gp

21and 2 120583g120583L human colorectal cancer homogenate

M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)

Figure 7 Western blot analysis of clone number 29 (a) 1 primary antibody crude cell extract of pComb3 clone 2 primary antibody crudecell extract of number 29 clone 3 primary antibody mouse anti-His antibody 1 2 and 3 antigen P-gp

21protein secondary antibody ALP-

labeled horse anti-mouse IgG antibody (b) 1 Antigen BSA 2 antigen 15 kDa peptide both 3 and 4 antigen P-gp21protein 1 2 3 and 4

primary antibody purified Fab secondary antibody HRP-labeled goat anti-mouse IgG antibody

resistance Among themmultidrug resistance (MDR)may bethemajor obstacle affecting the effectiveness of the treatmentMDR occurrence varied among patients of whom someare naturally resistant to multiple drug treatments (intrinsicresistance) while some may be induced by single drugtreatment (acquired resistance) [15] Due to cross-resistanceMDR limits the therapeutic efficacy of many important anti-tumor drugs In this study we engineered a novel antihumancolorectal cancerMDRgene (P-gp) Fabmonoclonal antibodyusing the spleen tissue from the immunized mice Becauseof the small size without Fc regions Fab antibody showedhigh specificity and sensitivity in detection of colorectaltumors with the MDR suggesting that Fab antibody may bedeveloped as a novel screening tool for personalized therapyof the patients who have colon cancer

Overexpression of P-gp is normally considered as themechanism of the cytotoxic action of anticancer drugs [1 16]The P-gp protein has been developed as a biomarker used

for detection of multiple drug resistance and monitoringchemotherapy in a number of studies [8 17 18]

A number of studies showed that complete IgG has someside effects due to FC fragments The complete IgG has along serum half-life resulting in poor contrast during theimaging process additionally inappropriate activation ofthe Fc receptor induces the cross-reactivity giving rise toassociated false positive signals especially when it is usedfor FCM [9 12 19] The size of the antibody moleculesis an important parameter in analysis of pharmacokineticsand biodistribution Compared to full-sized antibodies (withmolecular weight 150 kDa) which have shown slow pen-etration of the solid tumors and nonuniform distributionFab fragments show rapid penetration to solid tumor andpoor tumor retention Meanwhile due to the poor tissuepenetration rate and the defeats of the cross-reactivity thefull-sized antibody is seldom used in medical applications[11 12 19 20]


M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)

Figure 8 Verification of Fab soluble expression vector by Not I digestion (a) M marker DL2000 lanes 1-2 number 1 and number 2 cloneswere digested by Spe INhe I for deleting gIII fragment (b) M marker DL10000 lane 1 recombinant with no gIII deletion digested by Not Ilane 2 recombinant after gIII deletion digested by Not I

(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)

Figure 9 Fab expression and purification (a) SDS-PAGE 1 pComb3 clone was induced by 05mM IPTG at 28∘C for 8 h 2 number 29 wasnot induced 3 number 29 was induced by 05mM IPTG at 28∘C for 6 h 4 number 29 was induced by 05mM IPTG at 28∘C for 8 h (b)SDS-PAGE 1 sample before passing HiTrap Protein L column 2 sample after passing HiTrap Protein L column 3 the washout of the HiTrapProtein L column with binding buffer 4 the washout of the HiTrap Protein L column with sodium acetate buffer (pH 23)

Intriguingly although the difference in nucleotide se-quence between the P-gp

21recombinant and the P-gp gene

occurs in two sites the antigenicity of three epitopes of P-gp21was not affectedThus the Fabmonoclonal antibodywas

generated successfully from clone number 29A phage-displayed ldquolibraryrdquo is a heterogeneous mixture

of such phage clones each carrying a different foreign DNAinsert and therefore displaying a different peptide on its sur-face The phage antibody library technique is characterized

by a large capacity and maximal diversity of antibody librarywhich is the primary guarantee for obtaining high bioactivityFab fragments [13 21]

The quantity the diversity and the mature degrees of theantibody library and the size of the library capacity can beaffected by factors including (1) quality of RNA high qualityof total RNA (A260A280 gt 10) ensured the quality and thevolume of the library (2) maximal diversity it can be doneby tailoringmore degenerate primers (17 Fd-primer pairs and


0

100

200

300

400

500

600

700

RFU

The exact epitope recognized by Fab 29

Negative sampleFab sample

BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA

Figure 10 Identification of the exact epitope recognized by the anti-P-gp Fab by indirect immunofluorescence

0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU

Log10 concentration (ngmL)

Fab 29 (16-peptide-BSA)

Figure 11 Binding activity assay of anti-P-gp Fab to 16-peptide-BSAand BSA by using indirect immunofluorescence

18 120581-primer pairs 2 120582-primer pairs) from several donors andsimultaneous construction of both the light chain library andthe Fd library and (3) cloning strategy high efficiency ofcloning and bacterial transformation are the key to achieve aFab library attaining more than 106 cfu of the recombinants

In order to achieve a specific antibody against P-gp fromthe antibody library with low Fab recombinant frequencythe new panning strategy was used which is to increase thewashing time gradually during the five-round panning [11]Phagemids may be enriched effectively during panning andthe specificity of the Fab displayed on the phagemid may beincreased Another important consideration for obtaining thehigh bioactivity phagemids is to use high purity of the P-gp

21

antigen

Overexpression of the P-gp protein also prevents stemcell from differentiation leading to proliferation and ampli-fication of the cell repertoire The P-gp-caused MDR existsfrequently in the residual tumor cells after chemotherapy andthe tumor stem cells inducing tumor metastasis [22 23]The drug-transporting property of stem cells conferred bythe P-gp has been used as an important marker in isolationand analysis of cancer stem cells [24] Understanding themechanisms of cancer stem cell resistance to chemotherapymight therefore lead to discovery of a new therapeuticstrategy for therapy of cancer [25 26] Therefore detectionof P-gp expression in tumor cells and suppression of the P-gp-mediated active efflux of chemotherapeutic drugs fromthe tumor cells may be used as an index for evaluation ofchemotherapy [8 27ndash29]

Improvement of the MDR sensitivity by targeting the P-gp has been extensively used as a strategy for therapy ofcancer for more than 2 decades Many agents modulatingthe function of the P-gp have been identified includingcalcium channel blockers calmodulin antagonists steroidalagents protein kinase C inhibitors immunosuppressivedrugs antibiotics and surfactants [25 28 30] However sideeffects of these agents often occur because of their lowbindingaffinities One of the critical points to be addressed is howto achieve clinically effective doses of these chemosensitizersin circulation without producing dose limited side effectsThe use of the anti-P-gp MAbs as MDR-reversing agentsmay be the optimal approach to overcome MDR [31ndash33]Moreover targeting the P-gp by small-molecule compoundsandor antibodies is an effective strategy to overcome MDRin cancer [33 34]

5 Conclusion

The peptide sized 21 kDa covering the P-gp transmembraneregion was first prepared by us And both anti-human col-orectal cancer P-gp

21Fab antibody and its gene sequence have

been successfully obtained by phage display technology Wefound that the Fab antibody possessed better avidity eitherwith human colorectal cancer or transmembrane domains ofhuman P-gp

21 After having been purified by HiTrap Protein

L the concentration of Fab antibody reaches 150mgL Theexact epitope of human P-gp which the Fab monoclonalantibody recognized by indirect immunofluorescence wasALKDKKELEGSGKIAT Development of the specific Fabantibody against P-gp may provide an optimal strategy foreffective administration of personalized therapy of colorectalcancer and an effective screening tool for intervention ofmultidrug resistance

Conflict of Interests

Theauthors of the paper do not have a direct financial relationwith the commercial identity mentioned in their paper

Acknowledgments

This research was supported by a Grant of National Technol-ogy RampDProgram for the 11th five-year plan (2009BAK61B04)


The results described above had been submitted two patentapplications (2012103524391 20121037925200)

References

[1] P D W Eckford and F J Sharom ldquoABC efflux pump-basedresistance to chemotherapy drugsrdquo Chemical Reviews vol 109no 7 pp 2989ndash3011 2009

[2] F J Sharom ldquoThe P-glycoproteinmultidrug transporterrdquo Essaysin Biochemistry vol 50 no 1 pp 161ndash178 2011

[3] T W Loo and D M Clarke ldquoDetermining the dimensionsof the drug-binding domain of human p-glycoprotein usingthiol cross-linking compounds as molecular rulersrdquo Journal ofBiological Chemistry vol 276 no 40 pp 36877ndash36880 2001

[4] C Oswald I B Holland and L Schmitt ldquoThe motor domainsof ABC-transporters what can structures tell usrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 372 no 6 pp385ndash399 2006

[5] U A Germann ldquoP-glycoproteinmdasha mediator of multidrugresistance in tumour cellsrdquo European Journal of Cancer vol 32no 6 pp 927ndash944 1996

[6] H S L Chan P S Thorner G Haddad and V LingldquoImmunohistochemical detection of P-glycoprotein prognosticcorrelation in soft tissue sarcoma of childhoodrdquo Journal ofClinical Oncology vol 8 no 4 pp 689ndash704 1990

[7] R Clarke F Leonessa and B Trock ldquoMultidrug resistanceP-glycoprotein and breast cancer review and meta-analysisrdquoSeminars in Oncology vol 32 supplement 2 pp S9ndashS15 2005

[8] S Chiampanichayakul S Anuchapreeda N ChruewkamlowK Mahasongkram P Thanaratanakorn and W KasinrerkldquoProduction of monoclonal antibodies to P-glycoprotein itsapplication in detection of soluble and surface P-glycoproteinof leukemia patientsrdquo International Journal of Hematology vol92 no 2 pp 326ndash333 2010

[9] S Song B Li L Wang et al ldquoA cancer protein microarrayplatformusing antibody fragments and its clinical applicationsrdquoMolecular BioSystems vol 3 no 2 pp 151ndash158 2007

[10] L Friedrich S Stangl H Hahne et al ldquoBacterial productionand functional characterization of the Fab fragment of themurine IgG1120582 monoclonal antibody cmHsp701 a reagent fortumour diagnosticsrdquo Protein Engineering Design and Selectionvol 23 no 4 pp 161ndash168 2010

[11] P Holliger and P J Hudson ldquoEngineered antibody fragmentsand the rise of single domainsrdquo Nature Biotechnology vol 23no 9 pp 1126ndash1136 2005

[12] T Olafsen and A M Wu ldquoAntibody vectors for imagingrdquoSeminars in Nuclear Medicine vol 40 no 3 pp 167ndash181 2010

[13] M A Clark ldquoStandard protocols for the construction of Fablibrariesrdquo Methods in Molecular Biology vol 178 pp 39ndash582002

[14] DW J Coomber ldquoPanning of antibody phage-display librariesStandard protocolsrdquoMethods in Molecular Biology vol 178 pp133ndash145 2002

[15] S Eyal J G Lamb M Smith-Yockman et al ldquoThe antiepilepticand anticancer agent valproic acid induces P-glycoprotein inhuman tumour cell lines and in rat liverrdquo British Journal ofPharmacology vol 149 no 3 pp 250ndash260 2006

[16] M J Smyth E Krasovskis V R Sutton and R W JohnstoneldquoThe drug efflux protein P-glycoprotein additionally protectsdrug-resistant tumor cells from multiple forms of caspase-dependent apoptosisrdquo Proceedings of the National Academy of

Sciences of the United States of America vol 95 no 12 pp 7024ndash7029 1998

[17] Z P Pavelic J Reising L Pavelic D J Kelley P J Stambrookand J L Gluckman ldquoDetection of P-glycoprotein with fourmonoclonal antibodies in normal and tumor tissuesrdquo Archivesof Otolaryngology vol 119 no 7 pp 753ndash757 1993

[18] G L Scheffer M Kool M Heijn et al ldquoSpecific detectionof multidrug resistance proteins MRP1 MRP2 MRP3 MRP5MDR3 P-glycoprotein with a panel of monoclonal antibodiesrdquoCancer Research vol 60 no 18 pp 5269ndash5277 2000

[19] X Lin J Xie and X Chen ldquoProtein-based tumor molecularimaging probesrdquoAmino Acids vol 41 no 5 pp 1013ndash1036 2011

[20] R A Beckman L M Weiner and H M Davis ldquoAntibodyconstructs in cancer therapy protein engineering strategies toimprove exposure in solid tumorsrdquo Cancer vol 109 no 2 pp170ndash179 2007

[21] G Rojas H Lamdan S Padron Y Munoz M Ayala andJ V Gavilondo ldquoEfficient construction of a highly usefulphage-displayed human antibody repertoirerdquo Biochemical andBiophysical Research Communications vol 336 no 4 pp 1207ndash1213 2005

[22] PM Chaudhary and I B Roninson ldquoExpression and activity ofP-glycoprotein a multidrug efflux pump in human hematopoi-etic stem cellsrdquo Cell vol 66 no 1 pp 85ndash94 1991

[23] M Dean T Fojo and S Bates ldquoTumour stem cells and drugresistancerdquo Nature Reviews Cancer vol 5 no 4 pp 275ndash2842005

[24] TKondo ldquoStem cell-like cancer cells in cancer cell linesrdquoCancerBiomarkers vol 3 no 4-5 pp 245ndash250 2007

[25] R Krishna and L D Mayer ldquoMultidrug resistance (MDR) incancerMechanisms reversal using modulators of MDR and therole ofMDRmodulators in influencing the pharmacokinetics ofanticancer drugsrdquo European Journal of Pharmaceutical Sciencesvol 11 no 4 pp 265ndash283 2000

[26] D Dingli and F Michor ldquoSuccessful therapy must eradicatecancer stem cellsrdquo Stem Cells vol 24 no 12 pp 2603ndash26102006

[27] X Li J-P Li H-Y Yuan et al ldquoRecent advances in P-glycoprotein-mediated multidrug resistance reversal mecha-nismsrdquo Methods and Findings in Experimental and ClinicalPharmacology vol 29 no 9 pp 607ndash617 2007

[28] H Yuan X Li J Wu et al ldquoStrategies to overcome or circum-vent P-Glycoprotein mediated multidrug resistancerdquo CurrentMedicinal Chemistry vol 15 no 5 pp 470ndash476 2008

[29] J Wang H Wang L Zhao et al ldquoDown-regulation of P-glycoprotein is associated with resistance to cisplatin and VP-16in human lung cancer cell linesrdquo Anticancer Research vol 30no 9 pp 3593ndash3598 2010

[30] HThomas andHM Coley ldquoOvercomingmultidrug resistancein cancer an update on the clinical strategy of inhibiting P-glycoproteinrdquo Cancer Control vol 10 no 2 pp 159ndash165 2003

[31] E B Mechetner and I B Roninson ldquoEfficient inhibition of P-glycoprotein-mediatedmultidrug resistance with amonoclonalantibodyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 89 no 13 pp 5824ndash5828 1992

[32] T Iwahashi E Okochi K Ariyoshi et al ldquoSpecific targetingand killing activities of anti-P-glycoprotein monoclonal anti-body MRK16 directed against intrinsically multidrug-resistanthuman colorectal carcinoma cell lines in the nude mousemodelrdquo Cancer Research vol 53 no 22 pp 5475ndash5482 1993


[33] P Iangcharoen W Punfa S Yodkeeree et al ldquoAnti-P-glycoprotein conjugated nanoparticles for targeting drug deliv-ery in cancer treatmentrdquo Archives of Pharmacal Research vol34 no 10 pp 1679ndash1689 2011

[34] R Li R Wu L Zhao M Wu L Yang and H Zou ldquoP-glycoprotein antibody functionalized carbon nanotube over-comes the multidrug resistance of human leukemia cellsrdquo ACSNano vol 4 no 3 pp 1399ndash1408 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


immunohistochemical method (IHCA) and flow cytometry(FCM) depend on the reaction of the P-gp protein to itscorresponding monoclonal antibody Nevertheless use ofthe intact monoclonal antibodies may provide false positivesignals in detection of the tumor cells since the Fc fragmentsof IgG may bind to the Fc receptors on the surface of normalcells as well Thus use of the Fab antibody may be superiorto reducing the false signal resulting from the intact antibody[9 10] Meanwhile Fab is smaller than IgG in size facilitatingtrafficking of the antigen across cell membrane and mayprovidemore information than the intact antibody during theICHA process [11 12]

In this communication we constructed and characterizedthe phage-displayed mouse Fab against human MDR1P-gp

2 Materials and Methods

21 Animals The study was conducted in accordance withnational guidelines for care and use of animals All micewere purchased by the DalianMedical University LaboratoryAnimal Center (approval number LA 2009-008)The animalsused in the study were age-matched mature BALBc femalemouse weighing approximately 18ndash22 g

22 Construction of Phage-Displayed Mouse Fab AntibodyLibrary against Human Colorectal Cancer MDR1P-gp SixBALBC female mice were injected subcutaneously (02mL10 g) with 05mgmL human colorectal cancer homogenateemulsified in complete Freundrsquos adjuvant (CFA) The samecolorectal cancer homogenate and aliquot of Freundrsquos incom-plete adjuvant were subcutaneously injected on the seventhand fourteenth days respectively Seven days after the lastimmune blood samples were collected and used for deter-mination of the antibody titer by ELISA When the antibodytiter reached above 1 512 the spleens of the mice wereremoved immediately frozen by liquid nitrogen and usedfor extraction of total RNA Specific cDNA was synthesizedfrom RNA samples with random primer using the RevertAidFirst Strand cDNA Synthesis Kit (Fermentas) according tothemanufacturerrsquos instructionsThe Fd chain Lambda chainand Kappa chain were then synthesized from the cDNAwith 37 pairs of primers which were designed to annealwith mouse FR1 and CH

1or CL regions with high fidelity

Taq DNA polymerase (Fermentas USA) respectively PCRamplification was performed for 35 cycles (1min at 94∘C1min at 55∘C and 1min at 72∘C) in a Thermal Cycler S100(Bio-Rad CA USA)

PCR products were pooled into independent collectionsof Fd chain 120581 chain and 120582 light chain and purified by usingan AxyPre DNA Gel Extraction Kit (Axygen CA USA)The recombinants with Fd were performed according tothe method described as ldquoAntibody Phage Display Methodsand Protocolsrdquo [13] The recombinants with light chain andpGEM-T were firstly digested by Xba I and Sac I (Fermen-tas USA) then ligated into the Fd-pComb3 library andtransfected into E coli XL1-Blue cells (Stratagene CA USA)repeatedly to obtain the Fab library (gt106 CFU)The presenceand size of the inserts were further confirmed sequentiallywith Xho ISpe I and Xba ISac I

23 Cloning and Expression of the P-gp TransmembraneRegion A gene encoding 185 amino acids covering P-gptransmembrane region was isolated from the colon can-cer tissues and amplified by PCR using the sense primer51015840 CCGGAATTCCTCACCAAGCGGCTCCGAT 31015840 andthe antisense primer 51015840 CCGCTCGAGGAGTTTATGTGC-CACCAAGTAG 31015840 and then cloned into a pET28a (+)vector The recombinant was further verified by sequencingThe positive recombinants were selected and amplified byadding 10mM IPTG for 6 h at 30∘C Cell pellets werecollected by centrifugation at 14006timesg for 3min Proteinwasextracted from the cell pellets by using the nickel column (GEHealthcare CA USA) The purified P-gp

21was verified by

Western blot with the His-tag monoclonal antibody (1 2000)(Protein Tech CA USA)

24 Phage Display and Screening of the Clones Phagemid res-cuing and the Fab displayed library screeningwere conductedaccording to the protocol described [14] Phage particlesexposing antibody fragments were rescued from the Fablibrary with helper phage with VCSM13 helper phage (26 times1011 pfumL) on a 20mL scale The resulting phage mixtureswere used for binders selection on Nunc-Immuno MaxiSorpflat-bottomedmicrotiter plates (NuncDenmark) coatedwith49 120583g100 120583L of P-gp

21dissolved in bicarbonate buffer (pH

96) per well overnight at 4∘C Coated plates were incubatedin 3 BSA in PBS 300 120583L per well for 2 hr at 37∘C forblocking Then 200120583L Fab phage particles prepared abovewere incubated in microtiter plates for 1 h at RT Nonboundphages were washed away for 1 5 10 15 to 20 times perround with PBS containing 01 Tween 20The bound phageparticles were then eluted with 200120583L of elution buffer (01MHCl-glycine pH 22) per well and immediately neutralizedwith 14 120583L of 2M Tris base solution Collected phages wereused to infect E coliXL1-Blue cells in an exponential state andamplified as described above Rescued phage particles wereused to start a new selection round in the same conditionsaccording to the same protocol as described above [14]The phagemid titer was checked by counting the colony-forming unit (CFU) of the phagemid infected by XL1-Bluecells just before and after each panning After the fifth roundof screening a total of 10 colonies were picked randomly andverified by a double digestion using enzymes Xba ISac I andXho ISpe I respectively

25 Further Verification of the Anti-P-gp Positive Clones byELISA The positive Fab colonies were picked to test theirP-gp21

recognition properties Afterwards aliquots (20120583L)of the positive clones were transferred to 10mL of LB-Amp-Tet and further cultured until their A600 reached the sameas 04 Soon after IPTG was added to a final concentrationof 005mM and kept shaking at 220 rpm at 28∘C for sixhours The supernatants of each clone after centrifugation at14006timesg for 2min at 4∘Cpellets were collected and sonicatedfor 10min for ELISA

Moiety of the purified P-gp21

(1120583g120583L) and human col-orectal cancer homogenate (2120583g120583L) was incubated in 50120583Lbicarbonate buffer (pH 96) at 4∘C overnight respectively












3 Results





5000

2000

1000

(bp) M 1

28S

18S

(a)

(bp) M 1 1918171615141312111098765432 20

750500

(b)

(bp) M 1 1615141312111098765432 17

750500

(c)









21


21protein was




(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(a)

(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(b)

(bp) M 1 1098765432 M 1998400 2998400 3998400 4998400 5998400 6998400 7998400 8998400 9998400

750500

10998400

(c)















same OD600
























(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)








72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)















4 Discussion



001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Results





5000

2000

1000

(bp) M 1

28S

18S

(a)

(bp) M 1 1918171615141312111098765432 20

750500

(b)

(bp) M 1 1615141312111098765432 17

750500

(c)









21


21protein was




(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(a)

(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(b)

(bp) M 1 1098765432 M 1998400 2998400 3998400 4998400 5998400 6998400 7998400 8998400 9998400

750500

10998400

(c)















same OD600
























(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)








72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)















4 Discussion



001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5000

2000

1000

(bp) M 1

28S

18S

(a)

(bp) M 1 1918171615141312111098765432 20

750500

(b)

(bp) M 1 1615141312111098765432 17

750500

(c)









21


21protein was




(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(a)

(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(b)

(bp) M 1 1098765432 M 1998400 2998400 3998400 4998400 5998400 6998400 7998400 8998400 9998400

750500

10998400

(c)















same OD600
























(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)








72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)















4 Discussion



001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(a)

(bp) M 1 1098765432 M

750500

19984002998400399840049984005998400699840079984008998400999840010

998400

(b)

(bp) M 1 1098765432 M 1998400 2998400 3998400 4998400 5998400 6998400 7998400 8998400 9998400

750500

10998400

(c)















same OD600
























(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)








72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)















4 Discussion



001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















(bp) M 1

2000

750

500

28S

18S

(a)

(bp) M 1 2

750

500

(b)

(bp) M 1 2

7000

4000

1000

500

(c)

(bp) M 1

7000

4000

1000

500

(d)








72

26

17

26

17

(kDa) 52 31M 4

(a)

(kDa) 52 31M 4

72

26

(b)

(kDa) 21

26

17

(c)















4 Discussion



001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


001020304050607

Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

OD405nm


(a)

0010203040506070809

OD405nm


Blan

k co

ntro

lN

egat

ive c

ontro

lN

umbe

r 3N

umbe

r 5N

umbe

r 8N

umbe

r 9N

umbe

r 10

Num

ber 1

1N

umbe

r 13

Num

ber 1

4N

umbe

r 15

Num

ber 1

9N

umbe

r 22

Num

ber 2

3N

umbe

r 24

Num

ber 2

7N

umbe

r 29

(b)




M 1 M 2 M 3

26kDa

(a)

1 2 3 4

26kDa

(b)










M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2

750500

(a)

100007000

4000

2000

1000

500

250

M 1 2

(b)


(kDa) M 1 2 3 4

130

100

70

55

40

35

25

15

(a)

(kDa) M 1 4 5 2 3

170130

9572

55

43

34

26

(b)










0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

100

200

300

400

500

600

700

RFU



BSA

lowast

10-peptide-BSA

12-peptide-BSA

16-peptide-BSA


0

50

100

150

200

250

300

350

400

450

0 1 2 3 4

BSA

RFU






21

antigen



5 Conclusion








Acknowledgments




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article characterization of human colorectal...

Documents