Skip to main content
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1966 Jun 1;123(6):1061–1081. doi: 10.1084/jem.123.6.1061

STUDIES ON HUMAN ANTIBODIES

IV. PURIFICATION AND PROPERTIES OF ANTI-A AND ANTI-B OBTAINED BY ABSORPTION AND ELUTION FROM INSOLUBLE BLOOD GROUP SUBSTANCES

Manuel E Kaplan 1, Elvin A Kabat 1
PMCID: PMC2138172  PMID: 4161310

Abstract

Insoluble blood group substances prepared by copolymerization of soluble blood group substances with N-carboxy-L-leucine anhydride were used to absorb blood group antibodies from two human, high-titered anti-A sera. After the absorbants were washed free of nonspecific serum proteins, blood group antibodies were eluted either with pH 3.62 acetate buffer, or at neutral pH with monosaccharide haptens of the A or B antigenic determinants (N-acetyl-D-galactosamine or D-galactose respectively). The purified anti-A antibodies were characterized, immunoelectrophoretically, as γM-, γA-, and γG-immunoglobulins. These were further separated into γM- and γG-fractions by gel filtration or density gradient centrifugation. Both γM- and one of the two γG-antibody fractions contained K and L light chain determinants; the remaining γG-fraction was comprised, almost totally, of type K molecules. Precipitability of the purified anti-A immunoglobulins by blood group A substance varied from 43 to 89%. The agglutinating activity per unit N of the isolated γG-anti-A was found to equal, in one case, and to exceed, in the second, that of the γM-antibodies from the same individuals. The marked differences between γM- and γG-antibody fractions in quantitative hapten inhibition studies were interpreted to mean that the antibody-combining site of the isolated eluted γG-anti-A was significantly larger than that of the eluted γM-anti-A. Whether these data connote differences in combining site size between entire immunoglobulin classes in an individual serum or simply reflect the properties of highly selected antibody populations cannot be stated at present.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BERNIER G. M., CEBRA J. J. POLYPEPTIDE CHAINS OF HUMAN GAMMA-GLOBULIN: CELLULAR LOCALIZATION BY FLUORESCENT ANTIBODY. Science. 1964 Jun 26;144(3626):1590–1591. doi: 10.1126/science.144.3626.1590. [DOI] [PubMed] [Google Scholar]
  2. Beychok S., Kabat E. A. Optical activity and conformation of carbohydrates. I. Optical rotatory dispersion studies on immunochemically reactive amino sugars and their glycosides, milk oligosaccharides, oligosaccharides of glucose, and blood group substances. Biochemistry. 1965 Dec;4(12):2565–2574. doi: 10.1021/bi00888a004. [DOI] [PubMed] [Google Scholar]
  3. COHEN S., PORTER R. B. STRUCTURE AND BIOLOGICAL ACTIVITY OF IMMUNOGLOBULINS. Adv Immunol. 1964;27:287–349. doi: 10.1016/s0065-2776(08)60710-5. [DOI] [PubMed] [Google Scholar]
  4. Campbell D. H., Luescher E., Lerman L. S. Immunologic Adsorbents: I. Isolation of Antibody by Means of a Cellulose-Protein Antigen. Proc Natl Acad Sci U S A. 1951 Sep;37(9):575–578. doi: 10.1073/pnas.37.9.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. FRANKLIN E. C., FUDENBERG H. H. ANTIGENIC HETEROGENEITY OF HUMAN RH ANTIBODIES, RHEUMATOID FACTORS, AND COLD AGGLUTININS. Arch Biochem Biophys. 1964 Mar;104:433–437. doi: 10.1016/0003-9861(64)90486-2. [DOI] [PubMed] [Google Scholar]
  6. GRABAR P., WILLIAMS C. A. Méthode permettant l'étude conjuguée des proprietés électrophorétiques et immunochimiques d'un mélange de protéines; application au sérum sanguin. Biochim Biophys Acta. 1953 Jan;10(1):193–194. doi: 10.1016/0006-3002(53)90233-9. [DOI] [PubMed] [Google Scholar]
  7. GREENBURY C. L., MOORE D. H., NUNN L. A. REACTION OF 7S AND 19S COMPONENTS OF IMMUNE RABBIT ANTISERA WITH HUMAN GROUP A AND AB RED CELLS. Immunology. 1963 Sep;6:421–433. [PMC free article] [PubMed] [Google Scholar]
  8. HALBERT S. P., SWICK L., SONN C. The use of precipitin analysis in agar for the study of human streptococcal infections. II. Ouchterlony and Oakley technics. J Exp Med. 1955 May 1;101(5):557–576. doi: 10.1084/jem.101.5.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ishizaka K., Ishizaka T., Lee E. H., Fudenberg H. Immunochemical properties of human gamma-A isohemagglutinin. I. Comparisons with gamma-G and gamma-M-globulin antibodies. J Immunol. 1965 Aug;95(2):197–208. [PubMed] [Google Scholar]
  10. KOCHWA S., ROSENFIELD R. E. IMMUNOCHEMICAL STUDIES OF THE RH SYSTEM. I. ISOLATION AND CHARACTERIZATION OF ANTIBODIES. J Immunol. 1964 May;92:682–692. [PubMed] [Google Scholar]
  11. Kunkel H. G., Mannik M., Williams R. C. Individual Antigenic Specificity of Isolated Antibodies. Science. 1963 Jun 14;140(3572):1218–1219. doi: 10.1126/science.140.3572.1218. [DOI] [PubMed] [Google Scholar]
  12. LEDDY J. P., BAKEMEIER R. F. STRUCTURAL ASPECTS OF HUMAN ERYTHROCYTE AUTOANTIBODIES. I. L CHAIN TYPES AND ELECTROPHORETIC DISPERSION. J Exp Med. 1965 Jan 1;121:1–17. doi: 10.1084/jem.121.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lloyd K. O., Kabat E. A., Layug E. J., Gruezo F. Immunochemical studies on blood groups. XXXIV. Structures of some oligosaccharides produced by alkaline degradation of blood group A, B, and H substances. Biochemistry. 1966 May;5(5):1489–1501. doi: 10.1021/bi00869a007. [DOI] [PubMed] [Google Scholar]
  14. Lloyd K. O., Kabat E. A., Rosenfield R. E. Immunochemical studies on blood groups. XXXV. The activity of fucose-containing oligosaccharides isolated from blood group A, B, and H substances by alkaline degradation. Biochemistry. 1966 May;5(5):1502–1507. doi: 10.1021/bi00869a008. [DOI] [PubMed] [Google Scholar]
  15. MANNIK M., KUNKEL H. G. LOCALIZATION OF ANTIBODIES IN GROUP I AND GROUP II GAMMA-GLOBULINS. J Exp Med. 1963 Nov 1;118:817–826. doi: 10.1084/jem.118.5.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. NOSSAL G. J., SZENBERG A., ADA G. L., AUSTIN C. M. SINGLE CELL STUDIES ON 19S ANTIBODY PRODUCTION. J Exp Med. 1964 Mar 1;119:485–502. doi: 10.1084/jem.119.3.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. OUDIN J. B. Specific precipitation in gels and its application to immunochemical analysis. Methods Med Res. 1952;5:335–378. [PubMed] [Google Scholar]
  18. Onoue K., Yagi Y., Grossberg A. L., Pressman D. Number of binding sites of rabbit macroglobulin antibody and its subunits. Immunochemistry. 1965 Dec;2(4):401–415. doi: 10.1016/0019-2791(65)90039-x. [DOI] [PubMed] [Google Scholar]
  19. RAWSON A. J., ABELSON N. M. STUDIES OF BLOOD GROUP ANTIBODIES. VI. THE BLOOD GROUP ISOANTIBODY ACTIVITY OF GAMMA1A-GLOBULIN. J Immunol. 1964 Aug;93:192–198. [PubMed] [Google Scholar]
  20. ROSEVEAR J. W., SMITH E. L. Glycopeptides. I. Isolation and properties of glycopeptides from a fraction of human gamma-globulin. J Biol Chem. 1961 Feb;236:425–435. [PubMed] [Google Scholar]
  21. ROWE D. S., FAHEY J. L. A NEW CLASS OF HUMAN IMMUNOGLOBULINS. I. A UNIQUE MYELOMA PROTEIN. J Exp Med. 1965 Jan 1;121:171–184. doi: 10.1084/jem.121.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. SCHIFFMAN G., HOWE C. THE SPECIFICITY OF BLOOD GROUP A-B CROSS-REACTING ANTIBODY. J Immunol. 1965 Feb;94:197–204. [PubMed] [Google Scholar]
  23. SCHIFFMAN G., KABAT E. A., THOMPSON W. IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS. XXX. CLEAVAGE OF A, B, AND H BLOOD-GROUP SUBSTANCES BY ALKALI. Biochemistry. 1964 Jan;3:113–120. doi: 10.1021/bi00889a018. [DOI] [PubMed] [Google Scholar]
  24. SCHLOSSMAN S. F., KABAT E. A. Specific fractionation of a population of antidextran molecules with combining sites of various sizes. J Exp Med. 1962 Oct 1;116:535–552. doi: 10.1084/jem.116.4.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. WATKINS W. M., MORGAN W. T. Inhibition by simple sugars of enzymes which decompose the blood-group substances. Nature. 1955 Apr 16;175(4459):676–677. doi: 10.1038/175676a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES