THE USE OF FILTER PAPER CHROMATOGRAMS TO DEMONSTRATEANTIGEN-ANTIBODY REACTIONS

DONALD H. SPALDING' AND T. G. METCALFDepartment of Bacteriology, University of Kansas, Lawrence, Kansas

Received for publication February 4, 1954

The interaction of antigen and antibody invitro is customarily conducted in a liquid or semi-liquid environment. Demonstrable reactions mayoccur in the form of different serological phe-nomena depending largely on the physico-chemical forces involved and the circumstancessurrounding the admixture of antigen andantibody.The use of filter paper by Castaneda (1950) to

demonstrate the presence of a Brucella antigen-antibody system introduced a technique in whichpaper and not a serological test tube constitutedthe milieu in which the reaction took place. Thetechnique featured the use of a stained Brucellaantigen placed on the surface of filter paperwhich was observed to resist the washing effectof a stream of isotonic saline in the presence ofBrucella immune serum. The phenomenon whichtook place in less than one minute was called"surface fixation" and was not observed to occurin the presence of normal or heterologous immuneserum. The observations made were confined tothe demonstration of this interaction betweenBrucella antigen and antibody.The present study was designed primarily to

explore the capacity of stained antigens preparedfrom known pathogenic strains of bacteria tofacilitate by means of paper chromatograms thedetection and measurement of in vitro agglutininreactivity. A second consideration concerned thepossible extension of the technique to a practicalrole in diagnostic bacteriology.

MATERIAL AND METHODS

Antigen preparation. Suspensions were pre-pared and stained from cultures of Brucellaabortus (strain A-19), Salmonella typhosa (strainATCC 6539), and Pasteurella tularenst8, (Japstrain). The bacteria chosen were selected fortheir importance as pathogens causing disease in

1 Present address: Biochemical ResearchLaboratory, Dow Chemical Co., Midland,Michigan.

man, the recognition of which frequently dependson the demonstration of agglutinins in the bloodstream.Tryptose agar was used to cultivate B. abortus,

nutrient agar for S. typhosa, and a glucose-cystine blood broth medium for P. tularensis.The cultures were harvested after 48 hours' in-cubation and inactivated in physiological salinecontaining 10 per cent formaldehyde.A distilled water suspension of bacteria was

prepared from the killed sedimented cells. Thedistilled water was used in the ratio of 5 ml foreach 1 ml of packed cell volume. A freshlyfiltered solution of hematoxylin (NationalAniline and Chemical Co.), prepared as Harris'modification of Delafield's hematoxylin, wasadded in the ratio of 1 ml for each 5 ml of thedistilled water suspension of bacteria. The stain-ing solution and the bacteria were allowed toremain in contact for 24 hours at room tempera-ture and the excess stain then removed by cen-trifuging the bacteria for a total of three times,using physiological saline to wash the cells. Thestained antigen finally was resuspended in threevolumes of diluent. The final suspension repre-sented 1 ml of packed cells for each 3 ml of diluentand contained approximately two billion micro-organisms per ml.

Toluidine blue (National Aniline and ChemicalCo.) was used during the study in addition tothe hematoxylin. The method of preparingtoluidine blue stained antigen was identical tothat described for the hematoxylin. Other dyes(crystal violet, methylene blue, and triphenyl-tetrazolium chloride) were tried and found to beunsuitable.

Antisera. The antisera were prepared in rabbits,a standard tube agglutination test being used todetermine the serum titer. The titers (last dilutionshowing perceptible agglutination) of the anti-sera used were: P. tularensis 1:2,560; B. abortus1:320; S. typhosa, "H" agglutinins, 1:2,560. Theantisera were stored in a dry ice chest in smalllots and used as needed.

160.

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PAPER CHROMATOGRAMS IN ANTIGEN-ANTIBODY REACTIONS

Filter paper. Thirty-one types of filter paper(Eaton & Dikeman) were compared for theirability to demonstrate the presence of agglutininsfor a stained Brucella antigen. The papers werecompared objectively by measuring the area ofwashed antigen in terms of square millimeters andconstructing an index ratio which compared thedegree of fixation experienced by the antigencontrol in contrast to that found for the homolo-gous and heterologous sera, respectively. Threefilter papers (E&D no. 5, 609, 613) were selectedas the most suitable for the rapid and easy de-tection of a positive reaction. It was interestingto note that the physical characteristics of thethree papers, as supplied by the manufacturer,were quite similar. For example, the thickness ofthe papers varied between 0.006 and 0.008inches; the wet strength values2 were 4, 4.8, and5.5; and the rapidity values3 were 60, 60, and 70.Of the three papers, E&D no. 609 was usedthroughout the studies.

Experimental procedure. The colored antigenwas spotted on the filter paper, about one-halfinch from the edge, with the aid of a Kahnpipette drawn out to a fine capillary point at thetip and calibrated to deliver 0.001 ml. The serumto be examined was added then by means of a 4mm wire loop and the strip of paper immediatelysuspended in physiological saline (pH 7.0). Thepaper was allowed to remain in contact with thesaline for 15 minutes. At the end of this intervalthe saline solvent had ascended to a height of 1to 1.5 inches. Three controls were used in eachtest: (1) a cell control of antigen alone, (2)antigen plus normal serum, (3) antigen plusheterologous immune serum.The reproducibility of the technique was found

to be excellent. The delivery of a measured 0.001ml volume of antigen insured the deposition ofthis reagent in the same concentration at alltimes. The selection of a 4 mm wire loop foraddition of serum in preference to the use of amodified Kahn pipette capable of delivering ameasured 0.001 ml volume was based largely onthe greater ease and convenience of using theformer, having determined that the delivery of ameasured 0.001 ml volume of serum was followed

2 The relative bursting strength of wet paperunder standardized conditions.

3 The relative filtration rate of distilled waterunder standardized conditions.

C CA CN CT

Figure 1. The fixation of a stained Brucellaantigen by immune Brucella serum.C-Cell control.CA-Cells plus immune Brucella serum.CN-Cells plus normal rabbit serum.CT-Cells plus immune Salmonella typhosaserum.

by exactly the same results in the test as thoseobtained with the wire loop.Upon completion of the saline washing, the

developed paper chromatogram was allowed toair-dry and a permanent photographic record ofthe data obtained through the use of photographicreproduction equipment (Contoura Legal Model).

RESULTS

It was possible to demonstrate repeatedly, bymeans of the filter paper technique, the occur-rence of an antigen-antibody reaction for the B.abortus, S. typhosa, and P. tularensis systems. Atypical result is illustrated by figure 1 in which aB. abortus antigen resists the washing action ofphysiological saline in the presence of homologousantibody. The antigen, normal serum, and heter-ologous immune serum controls all failed to showa similar resistance to the saline wash, and theircolumns were observed to ascend the paper strip.The effect of varying the method of developmentof the chromatogram (ascending or descendingchromatography) failed to influence either thecourse or extent of the reaction.The specificity of the technique was investi-

gated by testing each antigen with several heter-ologous immune sera. The antisera used werethose mentioned previously and a serum pre-

19541 161

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

DONALD H. SPALDING AND T. G. METCALF

t C- l*th, ,, CAeL CA QCL CA10 2/0 40() 80 1ll'60 320 640

Figure 2. The fixation of a stained Brucella antigen by dilutions of immune Brucella serum.C-Cell control.CN-Cells plus normal rabbit serum.CA-Cells plus dilutions of immune Brucella serum.

C CiT CT CT CT CT C? CT CT CT CT CT CT10 20 40 80 160 320 640 12OO 2560 5120

Figutre 3. The fixation of a stained Salmonella typhosa antigen by dilutions of immune Salmonellatyphosa serum.C-Cell control.CN-Cells plus normal rabbit serum.CT-Cells plus dilutions of immune Salmonella typhosa serum.

pared against Proteus, strain OX-19. Fixation of saline. Starting with the highest serum dilution,antigen occurred only in the presence of the re- one 4 mm loopful of each dilution was depositedspective homologous serum. over each prepared spot of stained antigen andThe sensitivity of the filter paper technique for the paper developed in saline. The procedure

measurement of the in vitro reactivity of ag- followed thereafter was the same as givenglutinins was determined by titrations carried out previously.on the filter paper. Twofold dilutions of each The results of the titrations indicated a gradual,mmune serum were prepared in physiological loss by the immune serum of the power to fix

162 [ VOI,.1:08

l!;

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PAPER CHROMATOGRAMS IN ANTIGEN-ANTIBODY REACTIONS

C CN CT CT CT10 20

CT40

CT CT CT CT CT CTil CT80 160 320 640 1280 2560 5120

Figure 4. The fixation of a stained Pasteurella tularensis antigen by dilutions of immune Pasteurellatularensis serum.C-Cell control.CN-Cells plus normal rabbit serum.CT-Cells plus dilutions of immune Pasteurella tularensis serum.

TABLE 1

A comparison of the serum titers obtained by thefilter paper and standard tube agglutination

methods

TITER OB- TITER OB-TAINED BY TAINED BYSTANDARD STANDARD TITER

IDENTITY OF TTUBE AGGLU- TUBE AGGLU- OBTAINED

IMMUNE SERUMTINATION TINATION BY FILTERIMMUNESERUM METHOD METHOD PAPER

USING USING METHODUNSTAINED STAINEDANTIGEN ANTIGEN

Brucella ............. 640* 640* lOtSalmonella typhosa. 2,560 2,560 320Pasteurella tularensis 2,560 1, 280 40

* Reciprocal of highest dilution of serum caus-ing perceptible agglutination.

t Reciprocal of highest dilution of serum result-ing in demonstrable fixation of antigen.

antigen upon dilution of the serum. In figure 2 ahematoxylin stained Brucella antigen is seen tobe fixed by a 1:10 but not to an appreciabledegree by a 1:20 dilution of homologous immuneserum. In figure 3 a fixation end point of the S.typhosa antigen seems to be reached with the1:320 dilution of immune serum, and in figure 4a fixation end point of the P. tularensis antigen

seems to have been reached with the 1:40dilution of immune serum.A comparison of the fixation titer with the

conventional tube agglutination titer for eachimmune serum is given in table 1. With theexception of the P. tularensis stained antigen, thetiters obtained in the standard tube agglutinationtest were identical, regardless of whether stainedor unstained antigen was used. When aliquots ofthe same sera were used in the filter paper tech-nique, the fixation titer of each serum was con-siderably less than its corresponding tube ag-glutination titer. Under the conditions of the teststhe use of filter paper to measure the in vitroreactivity of the three agglutinins was shown to beincapable of attaining the same degree of sensi-tivity exhibited by the standard tube agglutina-tion method.During the course of the investigation the

question arose as to whether one was actuallyobserving the stained antigen flowing up thepaper or the stain itself. Microscopic examinationsof the ascending columns of completed chromat-ograms where fixation did not occur disclosedthe presence of stained cells which had moved up-wards on the paper. A typical observation ispresented in figure 5 as evidence of the presence

19541 163

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

DONALD H. SPALDING AND T. G. METCALF [VOL. 68

Figure 5. A chromatogram of stained cells of Brucella on the surface of filter paper. A few of thecells can be clearly distinguished, while many appear as dark areas. Filter paper fibers can be seen inthe background.

D DIIT DA2 DP

Figure 6. The chromatogram obtained with a

hematoxylin solution used in place of stainedcells.D-Dye control.DN-Dye plus normal rabbit serum.

DA-Dye plus immune Brucella serum.

DP-Dye plus immune Proteus, strain OX-19,serum.

of stained cells at a point halfway up the filterpaper strip. These observations indicated that inthe absence of antibody or with concentrationsinadequate to cause the fixation of antigen, the

stained cells ascended the paper strip when sub-jected to the washing action of physiologicsaline.Although the results obtained with the filter

paper technique failed to demonstrate the exist-ence of any basis for fixation other than antigen-antibody interaction, it was considered advisableto investigate the possibility that the dye itselfmight be fixed to a variable degree by the immuneserum. The usual technique of performing a testwas followed with the exception that the hema-toxylin dye was substituted in place of the stainedantigen. The results of these tests presented infigure 6 indicated that no fixation of the dye oc-curred under the conditions of the test.The interaction of antigen and antibody was

shown to be unaffected by an environmental pHrange of 3 to 8. Although the dark blue color ofthe hematoxylin dye eventually faded to a paleyellow as the hydrogen ion concentration was in-creased, no simultaneous impairment of antigenfixation by homologous immune serum could bedemonstrated.

Attention was directed to the method of prepa-ration of the stained antigen when it was shownthat an antigen spotted on filter paper in advanceof use and allowed to dry remained fixed at the

164

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

1'A4'l],Al CHItMMATOGRAMS IN ANTIGEN-ANTIBODY REACTIONS

C CN CA CP C CT? CA CP

Sa1lie Rabbit serum

C CUl CZ, CP

SucroseFiguire 7. A comparison of the results obtained with a stained antigen of Brucella suspendle(d in saline,

noirmal rabbit serum, an(l stucrose vehicles.C-Cell control.CN-Cells plus niormal rabbit serum.CA-Cells plus immune Brucella serum.CI'-Cells plus immune Proteuis OX-19 serum.

same position and failed to wash upon develop-ment with solvent. The use of a suspendingvehicle was considered which would serve toprotect the antigen from the harmful effects ac-companying drying. One and one-half per centsolutions of gum guaiac, gum acacia, and gumghatti; a one per cent solution of sodium carboxy-cellulose; solutions of saturated dextrin and glu-cose; a 70 per cent sucrose solution; andl finallynormal rabbit serum were compared for theirability to protect a stained Brucella antigen fromthe adverse effects of drying.

Three vehicles were found to be of interest.The glucose suspended antigen retained its abilityto wash freely on the paper in the absence ofhomologous antibody for approximately threeweeks, a substantial increase in stability. W7hileglucose was the only vehicle to confer such pIro-tection, both the normal rabbit serum and sucrosecontributed to an improvement in the repioduci-bility of the antigen control in the routine test.An example of the effect of normal rabbit serumand a 70 per cent sucrose solution, using aBrucella system, is shown in figure 7. The use ofthe normal serum vehicle seemed to leadl to themost consistent results, and consequently its useas a suspending medium for the bacterial antigenswas observed throughout the study.

DISCUSSION

The use of filter paper chromatograms for thedetection of antigen-antibody interactions wasexplored with the thought of critically evaluatingthe usefulness of this technique and its possibleextension to some piractical irole in diagnosticbacteriology.The experimental procedure wrhich was used

at the outset of the study was patterne(d after themethod of Castaneda (1950) who was the firstto introduce this technique. As the study pro-gressed, however, with each stel) of the proceduresubjected to a careful scrutiny, it was possible toeffect a critical evaluation of the entire technique,and as a result of these observations the originalmethod was modified and improved. The twostaining solutions, hematoxylin and toluidineblue, were selected after other staining solutionshadl been tried and found to be less suitable. Theantigen suspensions were spotted on filter paperwith the aid of a calibrated pipette in preferenceto a wiIre loop because the use of the formerseemed to impart a greater degree of reproduci-bility to the technique. The choice of a filterpaper was made on an arbitrary basis from threepapers of equal w-orth only after an exhaustivesurvey of 31 types of paper had shown these

19594] 165

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

DO)ONALD H1. SPALDING ANI) T. G. TMETCALF

thlee papers to be superior. The use of physio-logical saline (pH 7.0) as the solvent for chro-matogram development was decided on when itw-as noted that the daily fluctuations in the degreeof fixation which accompanied the use of tapwater were eliminated. The replacement of salineas the antigen suspending vehicle by normalrabbit serum was made when the latter wasshown to reduce the degree of nonspecific fixationof the antigen control and simultaneously increasethe degree of differentiation possible between thecontrol and test systems.

ThrIough the use of filter paper chromatograms,it was possible not only to confirm the fixation of astained Brucella antigen by homologous serum asreporte(l by Castaneda, but also to extend thetechnique to include P. tularensis and S. typhosasystems. In each instance the interaction ofantigen and antibody, judging from the fixationof the stained antigen, was rapid. The chromat-ograms were read routinely after a 15 minuteinterval of development by solvent, but in themajority of cases the outcome of the individualtest usually could be forecast accuratelv after 2to 3 minutes of development.The specificity of the technique as determined

by the antigen-antibody interactions was satis-factor-. No cross reactions were encounteredbetween any combinations of the antigen-antibody systems investigated.The sensitivitv of the reaction was pooIr when

the results wrere compared with those obtained inthe standar(d tube agglutination test. A possibleexplanation for the decreased sensitivity of thepaper chiomatograms might lie in the unusuallyconcentrated antigen suspensions which wereused to prodluce the desired visual intensity in thecompleted chromatogram. Sufficient antibodymight be plesent in undiluted high-titered serumfor complete agglutination and consequentfixation of all the antigen present. As the anti-body concentration is (lecireased, however, insuccessive twofold dilutions, a point would beattained at which insufficient antibody waspresent to sensitize all of the stained antigenparticles, and only partial agglutination wouldresult. There would exist a certain amount ofantigen remaining free to rise with the solvent.The extent of the rise in a given 15 minute de-velopment period might (lepend upon the ease ofescape of unsensitized antigen from the partiallyagglutinated mass.

In the case of the serum dilution at whichnumerous antigenic particles remained unsensi-tized, and aggregation of sensitized particles wasnot sufficient to impede the rise of the unsensitizedcells, the resulting visual effect would indicate nofixation of antigen when actually some fixationoccurred but remained undetected.The simplicity of the paper chromatogram

method, coupled with its reproducibility andspecificity, suggests the possibility of a practicalapplication. For example, the presence of bacterialagglutinins in man or animals might be deter-mined qualitatively in the space of a few minuteswith a minimum of equipment. The (letermina-tion could be made at once; there would be noneed to send or take a specimen of blood to alaboratory for analysis. Furthermore, if a courseof action (lepended on the outcome of the test, itcould be taken at once without delay.The finding that a glucose suspended Brucella

antigen retained partial reactivity after threeweeks of drying at room temperature was ofspecial interest when considering the abovepossibility. The measure of success achieved inthis (lirection gave hope that a stable antigenmight eventually be prepared which would retainits reactivity for indefinite periods of time afterbeing spotted on filter paper.

SUMMARY

The results of the present study were inter-pireted to indicate that the interaction of antigenand antibody on filter paper is comparable in allrespects except one to the same interaction car-ried out in a test tube. The sole exception involvedthe sensitivity of the filter paper method, thetiter of agglutinins detected by this means beingless than the titer of the same agglutinins deter-mined by the standard tube agglutination test.Through the use of the filter paper technique

the presence of agglutinins for stained antigens ofBrucella abortus, Pasteurella tuilarensis, andSalmonella typhosa was detected.A practical use for the filter paper technique

was l)roposed, and some of the circumstancessuriounding this use were discussed.

REFERENCECASTANEDA, AM. R. 1950 Surface fixation. A

new mnethod of detecting certain immunologi-cal reactions. Proc. Soc. Exptl. Biol. Med.,73, 46-49.

I16f, [VOI,. 68

on June 30, 2020 by guesthttp://jb.asm

.org/D

ownloaded from