The Immune-Adherence Activity of Normal Sera with Respect to Certain Particulate Antigens
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Vol. XL, No. 1 (February, 1959), wasised on 13.2.59. THE BRITISH JONAL OF EXPERIMENTAL PATHOLOGY VOL. XL APRIL, 1959 NO. 2 THE IMMUNE-ADHERENCE ACTIVITY OF NORMAL SERUM J. L. TURK From the London School of Hygiene and Tropical Medicine, Keppel Street, London, W.C.1 Received for publication August 22, 1958 A HEAT labile non-specific complement-fixing activity in normal serum was described by Mackie and Finkelstein (1930). A similar activity was described by Pillemer et al. (1954) which they called the properdin system. That part of the properdin system which is absorbed by zymosan and a wide range of poly- saccharides has been shown to account for some of the bactericidal and viricidal activity of normal serum (Pillemer et al., 1954 and 1955). Properdin appears to need Mg+ + ions and serum co-factors resembling the 4 components of comple- ment for its action. The immune-adherence phenomenon (Nelson, 1953), in which an antigen coated with its specific antibody and after fixing all 4 components of complement will adhere to the primate red cell, provides a sensitive test with which to re- explore the immunological activity of normal sera. Antibody to particulate antigens can be titrated by counting the numbers of red cells with adherent antigens, in immune-adherence reaction mixtures, under the phase contrast microscope. Nelson and Lebrun (1956) found that normal guinea-pig serum caused the immune-adherence of starch grains and Nelson and Kelsey (personal communi- cation) found the same true for zymosan. The present work describes the immune-adherence activity of normal sera with respect to certain particulate antigens. Two forms of activity are dis- tinguished, one specific and the other non-specific. The relation between the non-specific immune-adhering activity and the properdin system are discussed. MATERIALS AND METHODS Veronal buffered 8aline with added Ca++ and Mg++ was used throughout as diluent (Fulton and Dumbell, 1949). Antigens Amaranthus cruentu8 starch (Nelson and Lebrun, 1956) was used as an antigen in immune- adherence experiments, because its size in relation to that of red cells enables grains adhering 9 98 J. L. TURK to red corpuscles to be easily counted. Rice starch (B.D.H.) was used for absorbing sera. Zymosan was obtained from the Nutritional Biochemical Corporation, Cleveland, Ohio. Bacteria were grown on nutrient agar, killed at 600 for 1-hr. and washed 3 times in saline before use. Polys8acharides Amylose, dextran and laminarin were kindly given by Dr. W. Whelan of the Lister Institute, London, yeast mannan by Dr. D. H. Northcote of the Department of Biochemistry, University of Cambridge, and levan by Dr. M. Shilo of the Department of Microbiological Chemistry, the Hebrew University, Jerusalem. Inulin was obtained from George T. GOurr Ltd., and xylan from the Nutritional Biochemical Corporation. Sera All sera were stored at -700. All guinea-pig serum was pooled, each pool containing the sera from 6-8 animals. Samples of serum taken from newborn calves before suckling were kindly given by Dr. A. E. Pierce of the Agricultural Research Council's Animal Physio- logy Research Establishment, Babraham. Complement Undiluted guinea-pig serum for complement was absorbed twice at 40 with 50-100 mg. (dry weight) per ml. of the antigen to be used, and used in a dilution of 1/32. Horse serum was used as a source of complement in experiments on the immune-adherence activity of pre-suckling calf serum and was similarly absorbed before use. Erythrocytes Human group 0 blood was stored in Alsever's solution at 4'. The cells were washed three times in veronal buffered saline before use. Immune-adherence titrations Antigen in 0 5 ml. amounts (2 x 108 bacteria/ml., or 0 125 mg./ml. of starch or zymosan), 0-2 ml. of the serum to be tested, titrated out in doubling dilutions, and 0-2 ml. of absorbed serum to provide an excess of complement, were incubated in Kahn tubes at 370 for 20 min. in a water bath. Volumes of 0.1 ml. of 1-5 per cent washed group 0 human red cells were added and the tubes replaced in the water bath 1 hr. The end point of the titration was judged roughly by the haemagglutination patterns which formed at the bottom of the tubes after this time. The cellular deposit at the bottom of the tubes was then examined under the phase contrast microscope and the titre was taken as the reciprocal of the initial dilution of serum in the last tuibe that contained 20 per cent or more red cells with adherent antigenic particles. Bactericidal titrations These were carried out in M.R.C. plastic haemagglutination trays uising the technique of Nagington (1956). Absorption of sera Sera were absorbed twice at 40 with 50-100 mg. of bacteria (wet weight) per ml., or 100 mg./ml. of zymosan or polysaccharide, in 50 ml. flat-bottomed flasks on a horizontal rocking machine. All bacteria or water insoluible polysaccharides were washed 3 times in saline before being used for absorption. Fractionation of serum Sera were fractionated into euglobulin and pseudoglobulin fractions by dilution. The serum was diluted 1/10 in 0-005 M KH2PO4 at 0° for 30 min. The resulting precipitate was centrifuged off at 40 and washed 3 times in a phosphate solution pH 5-4 and ionic strength 0-02. It was redissolved in veronal buffered saline. The supernatant was brought back to isotonicity by the addition of 10 per cent NaCl, and the pH was brought back to 7-4 by the adc4itiort of 0-1 N NaOH, IMMUNE-ADHERENCE ACTIVITY OF SERUM 99 RESULTS The immune-adherence activity of normal guinea-pig, rabbit and human sera Table I shows the titres of immune-adherence activity, in the presence of an excess of complement, of normal guinea-pig serum (derived from a pool of 8 guinea-pigs weighing between 350 and 400 g.), normal rabbit serum (adult 12- month-old rabbit weighing 3-5 kg.), and an adult male human (aged 27 yr.). TABLE I.-Titre (1:) of Immune-adherence Activity of Normal Guinea-pig, Rabbit and Human Serum, in the Fresh Serum, Serum Heated at 550 for 20 min. and After Absorption with 0O1 g./ml. Zymosan twice at 40 Rabbit Guinea-pig (Adult male 3 kg. Human (Pool of 8) -1 yr old) (Adult male age 27 yr.) A- , .-- Heated Zymosan Heated Zymosan Heated Zymosan Fresh 55° absorbed Fresh 550 absorbed Fresh 550 absorbed Salm. typhi. 256 16 16 . 32 <8 <8 . 32 <8 16 Sh. flexneri 256 <8 16 . 128 128 128 . 128 8 64 Bact. coli . 128 <8 16 . 128 8 8 . 128 <8 64 V. el tor . 64 8 16 . 32 <8 <8. 64 8 32 Staph. aureu8 . 512 <8 16 . 128 16 8 64 8 32 Ery. rhusiopathiae 1024 1024 1024 . 32 <8 <8 32 <8 8 Starch . 512 < 8 <8 . 1024 1024 1024 . 64 <8 32 On absorbing the serum with 100 mg./ml. zymosan, or heating the serum at 550, there was a marked fall in the activity of the guinea-pig serum against all antigens tested except Erysipelothrix rhusiopathiae, the titre against which was unaffected by both these processes. There was a similar fall in the activity of rabbit serum, after heating and zymosan absorption, against all antigens except starch and Shigella ftexneri. The sera of some of the rabbits tested did however show up to a 4-fold fall in titre against these 2 antigens on zymosan absorption. In both guinea-pig and rabbit sera the fall in titre on zymosan absorption was very closely paralleled by the fall in titre on heating the sera at 550 for 20 min. However, on absorbing human serum with 100 mg./ml. zymosan twice for 1 hr. at 40, the immune-adherence titre fell only two-fold against all the antigens tested except Ery. rhusiopathiae, where the fall was four-fold; absorption was not increased at 22°. There was no relation between fall in immune-adherence titre after zymosan absorption and after heating at 550 for 20 min.; heating decreased the titre up to 16-fold, but zymosan absorption only 2-fold. Fractionation of the human serum (Table II) into euglobulin and pseudo- globulin showed that the immune-adherence activity of the serum against all the antigens tested except starch was divided between the 2 fractions. Activity against starch was completely in the euglobulin. Fractionation of the same serum after absorption with zvmosan showed a marked fall in the activity against all the antigens except starch in the euglobulin, but no fall in the activity in the pseudoglobulin. The activity against starch in the euglobulin showed a slight rise in titre after zymosan absorption. In other human sera there was a marked fall in the euglobulin titre against starch after absorption with zymosan and in one of them activity in the euglobulin against Sh. flexneri was not reduced Qn absorption of the serum with zymosan, 100 J. L. TURK TABLE II.-The Effect of Fractionation of Guinea-pig and Human Serum on the Titre (1:) Before and After Absorption with Zymosan Guinea-pig Human , ~~~~A . ,.A. Zymosan Zymosan Unabsorbed absorbed Unabsorbed absorbed Eu- Pseudo- Eu- Pseudo- Eu- Pseudo- Eu- Pseudo- globulin globulin globulin globulin globulin globulin globulin globulin Salm. typhi 64 <8 8 <8 . 8 64 <4 64 Sh. flexneri . 32 16 < 8 16 32 16 8 16 Bact. coli . 32 16 <8 16 128 32 4 32 V. el tor. 8 <8 <8 <8 16 64 4 64 Staph. aureus . 32 16 <8 16 . 16 16 <4 16 Ery. rhusiopathiae 16 32 <8 32 .