Augmenting Effect of Rheumatoid Sera in a Streptococcal Haemagglutination Test
Authors: N. R. Ling, H. J. Gibson
Journal: Annals of the Rheumatic Diseases (1957)

Culture filtrates of streptococci were shown by Rantz, Randall, and Zuckerman (1956) to contain a polysaccharide which attached itself to erythrocytes, preparing them for agglutination by human sera.This polysaccharide was produced by Gram-positive but not by Gram-negative organisms.Its distribu- tion suggested to us a possible relationship to an unidentified antigen responsible for the marked agglutination of streptococci and certain other Gram- positive organisms by sera of patients suffering from rheumatoid arthritis (Cecil, Nicholls, and Stainsby, 1930; Dawson, Olmstead, and Boots, 1932).A possible connexion was also suggested by a report that sera from patients suffering from some of the collagen group of diseases showed high titres in the streptococcal haemagglutination test (Rantz, 1953).The titres increased somewhat with age (Rantz, Zuckerman, and Randall, 1952), but apparently bore no relation to previous streptococcal infection ( Rantz, Zuckerman, and Randall, 1952; Harris and Harris, 1953).The ease with which this test may be performed made it possible for us to examine sera from a large number of patients of all clinical types.Although higher titres with sera from patients suffering from rheumatoid arthritis were not invari- able or specific, a tendency to high titres, which could not be accounted for on an age basis was noted, with occasional unexplained exceptionally high titres accompanied by a strongly positive Rose- Waaler test (Rose, Ragan, Pearce, and Lipman, 1948; Waaler, 1940).To account for this phenomenon it was postulated that the high titres obtained with such sera were due to the dual action of an antibody and a co-existing augmenting factor.Evidence in favour of this hypothesis is presented.* This paper was presented at a clinical meeting of the Hebe rden Society on December 14, 1956.Methods and Materials Human Erythrocytes of group 0 (usually Rh.-negative) were obtained from residues in recently returned blood- transfusion bottles, and were washed four times with saline before use.Similar titres were recorded in all test systems when the patient's own erythrocytes were employed.Rose-Waaler Tests were performed with human erythrocytes sensitized with one-quarter of a minimal agglutinating dose (j M.A.D.) of rabbit anti-human immune antibody exactly as previously described (Gibson and Ling, 1956) except that plastic agglutination trays were used.In almost every case the test was also carried out by the sheep cell differential test (Rose, Ragan, Pearce, and Lipman, 1948) and the results agreed with those given by the human cell system.Human Sera obtained from hospitals in the Bath Area were inactivated at 560 for 30 minutes before use.They were either fresh or had been stored at -15°C.Streptococcal Filtrates.-Type 12streptococci, supplied by the Public Health Laboratory, Bath, were grown in tryptose broth at pH 7 -2 for 4 days as recommended by Rantz, Randall, and Zuckerman (1956).The culture was acidified with a few drops of glacial acetic acid, brought to the boil, and the coarse coagulum of organisms filtered off.The filtrate was stored at -15°C. in 50-ml.quantities.For reference purposes, one culture filtrate was dialysed to remove inorganic salts and small organic molecules, taken to dryness under reduced pressure, desiccated, and ground to a fine powder.When recon- stituted with saline this preparation was shown to contain all the activity of the original broth.Polysaccharide-Coated Erythrocytes.-A"saturation dose" of culture filtrate was added to a 10 per cent.suspension of human erythrocytes in 0 15 M. pH 7 0 phosphate buffer.After incubation at 370 C. for 30 minutes the erythrocytes were centrifuged down, washed 111 copyright.