d R. J. Doyle ond 1. Cronholm Department of Microbiology University of Louisville School of Medicine Louisville, Kentucky 40202
A Model Antigen-Antibody System for Classroom Use
Studies of imnlunologic reactions form a n integral part of undergraduate and graduate level coursesin chemistry, biochemistry, and microbiology. Usuallv these courses are laboratorv-oriented and nroblems related to the procurement of antibodies and autigens are often encountered. Various antiserums are commerciauy available but costs are often prohibitive. I t is also, at times, not practical to attempt immunization procedures due to limitations on animal facilities. For those who might be interested in antigen-antibody interactions and have certain of the above limitations, we propose a model system which incorporates many facets of the immunologic reaction but does not require specific antiserum. The model system we have used for instructive purposes employs concanavalin A (a globulin isolated from jack bean seeds) as the model antibody and a restricted but readily available number of polysaccbarides as model antigens. Branched polysaccbarides which have non-reducing a-D-hexapyranosyl or 8-D-fructofuranosyl terminii (such as mannans, glycogens, and levans) form visible complexes with concanavalin A. Other polysaccharides such as galactans, amylases, arabinans, and linear dextraus are unreactive, demonstrating specificity of the precipitin reaction with concanavalin A. The reaction can be inhibited with monomeric or oligomeric sugars which have structural features similar to the reactive polysaccharide and, therefore, may be used as model haptens. The antigen-antibody-like character of the concanavaliu A-polysaccharide reaction has been demonstrated by Goldstein, et al. (1-7) and others (8-10).
The materials needed for classroom use of the concanavalin A-polysaccharide model system are commercially available and economical. The finely-ground Table 1 . Techniques Employed in the Study of Antigen Antibody Reactions and their Uses in the Concanavalin ACarbohydrate Model Svstem Reference Immunoadsorption (for the preparation of pure concanavalin A) Aear eeldiffusion (Ouchterlonvtechnioue)
precipitin reaction) Immunoelectrophoresis Hapten binding (for the determination of association constants and number of binding sites) Chemical modification of active binding site (simple experiment which shows the presence of tyrosine in the carbohydrate binding site oi concanavdin A ) Arthus-like reactions (experimentwhichshows that concanavalinA, similar to antibodies, can induce the hemorrhagic Arthus-like lesions in uiuo)
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(3) (6,8)
defatted jack bean meal provided by the Sigma Chemical Company, St. Louis, Ma., is an excellent source of concanavalin A. The polysaccharides and simple sugars are available from most chemical supply houses. No special apparatus is required other than that normally used in the study of immunologic interactions. Some of the major methods used in presenting the principles of the antigen-antibody reaction are summarized in the table. Each method has been applied successfully to the concanavalin A-carbohydrate system. References are given which provide complete experimental details with regard to the use of the concanavalin A-carbohydrate system. The literature cited will suggest many more experiments which could be undertaken by students interested in antigen-antibody reactions. For introductory or demonstration purposes we suggest the following experiments. Pipet 1 ml of concanavalin A (1-2 mg/ml) into 6-8 test tubes. Add 2 ml of commercial glycogen into each tube, varying the glycogen concentration from 0.05 mg in the first tube to 20-30 mg in the last tube. The tubes are mixed and incubated about 10 min and the resultant turbidity recorded on any suitable spectrophotometer. The samples are blanked against polysaccharide without added concanavalin A. A plot of absorbance versus glycogen concentration reveals the classical precipitin curve. Another series of tubes containing 1-2 mg of concanavalin A and 0.2 mg glycogen are mixed. To one tube is added 0.1 ml water, to the remaining tubes are added 0.1 ml of increasing quantities of wmannose or D-glucose (0.1-20 pmoles/O.l ml). The tubes are mixed, incubated 10 min and absorbances recorded. The per cent inhibition a t each sugar concentration is calculated and is plotted against log sugar concentration. This experiment shows the relative effectiveness of hapten inhibition by the various sugars. The precipitin reaction can also be demonstrated by agar gel diffusion. Place 0.1 ml of concanavalin A in the center well of an agar gel diffusionplate (2) and 0.1 ml of polysaccharide in the outer wells. To demonstrate specificity icjs convenient to use glycogen, soluble starch, yeast mannan, galactan, and arabinan in the outer wells. After an overnight incubation at room temperature, visible precipitin bands appear between the center well and the wells containing glycogen and yeast mannan. The agar gel precipitin bands slowly disappear when layered with haptens.
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Acknowledgment
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The authors were supported by grants from the University of Louisville (GRS-583403X) and by Public Health Service Postdoctoral Fellowship 5-F2-CA 19,083-02 from the National Cancer Institute.
Literalure Cited
(1) AanAw.iL, B. B. L., AND GOLDSTEIN, I. J., Bioehcm. J., 96, 23c (1965). (2) GOLDSTEIN, I. J., AND SO,L., Axh. Biocham. Biophgs., 111, 407 (1965). I. J., J. Biol. Chem., 242, 1617 (3) So, L., A N D GOLDSTRIN, (1967). 1. J., HOLLI:RMAN, C. W., AND SMITH,E. E., (4) GOLDSTEIN, Biochemistry, 4 , 876 (196.5). ( 5 ) So, L., AND GOLDSTEIN, I. J., Riochim. Biophys. A d a , 165,
398 (1968). (6) P o n ~ ~It. z , D., (19fiR). ~-.-.,
AND
GOLDSTEIN, I. J., Immunobgy, 14, 165
(7) GOLDSTEIN, I. J., HOLLERMAN, C. E., .AND ~ ~ I ~ R RJ.I M., CK, Biochim. Biophys. A d a , 97, 68 (1965). , KALII, A. J., AND LIYITSIU, A,, Biochim. Biophys. (8) Y a ~ wJ., Acla, 165, 303 (1968). R. J., AND ROAOLT, O., Life S C ~ C ~ L7C C (Part S , II), (9) DOYLE, 841 (1968). 110) KIND. L. S.. AND PETI:RSI:N. W. A,. Seimee. 160. 812
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