The Detection of Anti-Globulin Factors Utilizing Pre-Coated Latex Particles By JACQUES M. SINGER,CHARLES M. PLOTZAND ROSALIEGOLDBERG A modified serological procedure for the detection of anti-gamma globulin factors has been developed. This test employs polystyrene latex particles with a diameter of 0.25 microns. The particles are coated with gamma globulin and the excess protein in solution is removed by repeated washing. A solution of these gamma globulin coated particles is stable for at least one year, and yields more reproducible titers than those originally described for the latex agglutination test. Esseva disveloppate un modificate procedimento pro le detection de factores anti globulina gamma. Le test utilisa particulas de latex polystyrenic con un diametro de 0,25 micron. Le particulas es revestite de globulina gamma e le excess0 de proteina es eliminate per repetite ablutiones. Un solution de tal particulas revestite de globulina gamma es stabile durante a1 minus un anno. Le titros obtenite con ill0 es plus reproducible que illos describite pro le test original de agglutination a latex. U KIFORM SIZE LATEX PARTICLES 8000 A in diameter (Dow Chemical) have become widely used as particulate carriers of antigens for the detection of many antibodies or antibody-like substances.l The technic as originally applied to the serological measurement of rheumatoid, or antigamma globulin factors is known as the latex fixation or latex agglutination reaction (FII-LP) .* Bozsoky” recently used the FII-LP reaction as a prototype to emphasize the hazards in the reporting of results using a “standard test in different laboratories. Using standard lyophilized dried sera he asked 30 laboratories in 14 countries to perform FII-LP and sensitized sheep-cell (SSC) tests on a blindfold basis. Great variations in results were obtained and these proved to be due to small changes in technic. These changes included the use of latex particles from different commercial sources, variation in the size and concentration of the particles, differing concentrations of coating gamma globulin (ranging from 0.025 per cent to 5 per cent), inactilration of the sera and gamma globulin in some laboratories (in contradiction to standard technic), incubation periods ranging from none to 2 hours, and reading times from immediately to 19 hours later. It therefore was not surprising that a standard positive serum gave results from 1:32 to 1:20,000. This I ariability is particularly troublesome in research investigaticns and epidemiologic studies. It gives rise to obvious difficulties in the comparison of results obtained among various institutions at different times, if there has been a change in personnel. As a consequence, a modification of the latex fixation test has been developed utilizing latex particles 2300 A 2 200 A in ~ ~ _ _ _ _ _ _ From the Laboratory Division, Department of Microbiology, Mont4ore Hospital, Bronx, New York and the Department of Medicine, State Uniuwsity of New York, Downstate Medical Center, Brooklyn, N e w York. Aided in part b y grants No. AM 06052-01-02 from the National Institute of Arthritis and Atetaholic Diseases, National Institutes of Health untl by the N e w York Chapter of the Arthritis Foundation. 194 ARTHRITIS AND RHEUMATISM,VOL. 8, No. 2 (APRIL), 1965 DETECTION OF ANTI-GLOBULIN FACTORS 195 diameter, ( Lytron Monsanto Chemical). These particles are stable and are most effectively pre-coated with protein material. MATERIALSAND METHODS Muterials 1. Gamma Globulin preparations #I812 and #2015 were obtained from E. R. Squibb & Sons through the courtesy of the American Red Gross, Washington, D. C. 2. Fraction V Human Albumin was obtained from E. R. Squibb and Sons. 3. (3ycine-snline buffer at p H 9.6, ionic strength 0.24 (25 C.) was prepared liy adding 100 ml. of 1 N NaOH to 116 nil. of 2M glycine and bringing the volume to 1,000 ml. with distilled water, p H was adjusted to 9.6 and then 9 grams of sodium chloride were added to each 1,000 ml. of buffer. 4. Polystyrene Lytron particles were obtained through the courtesy of the Monsanto Chemical Company, Springfield, Mass. Lytron 601 (1700 A diameter) and Lytron 61518-1076 (serial #RF-2146) (2500 A diameter) were used. 5. Polystyrene Latex particles were prepared by Dr. John W. Vanderhoff of the Dow Chemical Co., Midland, Mich., who kindly provided the physico-chemical characteristics of each batch. Dow LS-040-A, LS-052-A, LS-463-E, LS-061-A, LS-063-A, LS-449-E, L6053-33-1, L-5309-19-3 and L-4849-44-2, varying in size from 500 A to 280.000 A, were used. 6. Merthiolate solution ( 1:10,000 in glycine-saline buffer) was added to prevent bacterial contamination. 7. Stabilaty i 5 defined as a uniform colloidal suspension with no visible agglutination or aggregation and with turbidity corresponding to the light transmission in a Coleman, Jr. Spectrophotometer of. a barium sulfate standard prepared by adding 3 ml. of 0.1 N barium chloride to 3 ml. of % N sulphuric acid. Unstable is defined as the presence of visible agglutination or aggregation and a light transmission of 100 p a cent. Partial Instability is defined as visible agglutination or aggregation and a light transmission betwem that of the barium sulfate standard and 100 per cent. Pre-Coating is defined as adsorption of gamma globulin on latex particles with subsequent washing until no more gamma globulin remains in the supernatant. Methods The general procedure was to add one ml. of latex suspension one ml. of solution of gamma globulin and to bring the total volume to 10 ml. with glycine-saline buffer at p H 9.6. The tubes were incubated for 1 hour at room temperature (approximately 23 C.) and centrifuged for 1 hour at 12,000 r.p.m. in a Servall refrigerated centrifuge. The particles cannot be sedimented readily at lower speeds. The supernatant was discarded and the sediment gently and gr'idually ( t o avoid flocculation and clumping) resuspended in one to two ml. of buffer, and the suspension brought to a final volume of 10 ml. This washing was repeated one more time. After the second washing the resultant stock solution was diluted with buffer to a 5 per cent transmission in a Coleman, Jr. Spectrophotometer, Model 11, at 650 mp. Each batch of coated latex particles was tested for agglutination activity using a standard rheumatoid arthritis serum with a 1:10,000 positive titer. The tubes were incubated in a water bath at 56 C. for 1%hours, refrigerated overnight at 4 C. and then centrifuged at 2,500 r.p.m. for 5 minutes. The resultant visible agglutination ranging from 1+ to 4+ was read with the naked eye. Agglutination titers were recorded as the reciprocal of the serum dilution. Selection of Particle Size. Thirteen batches of uniform-size latex particles ranging from 500 to 280,000 A in diameter were tested for stability in glycine-saline buffer at p H 9.6. Stability was determined utilizing uncoated and coated particles. Each batch was adjusted to the same surface area of 35 x 1018 A2 per ml. of latex suspension. Three con- 196 SINGER, PLOTZ, GOLDBERG Table 1.-A Comparison of the Stability of 0.25 Micron Lytron Particles Coated with Various Amounts of Gamma Globulin and Their Reactivity with a Rheumatoid Arthritis Serum Concentration of Gamma Globulin Added in mg./ml. 15 10.5 4.5 1.5 0.6 0.15 0.075 0.0375 0.0187 Ratio of Protein Added in mg. per em2of Particles Stability Particle Coated 4.3 x 10-3 3.0 x 10-3 1.3 10-3 4.3 10-4 1.7 10-4 4.3x 10-5 2.2 x 10-5 1.1x 10-5 5.5 x 10-0 stable stable stable stable stable stable unstable unstable unstable Agglutinating Titer 1/3200 1/3200 1/3200 1/3200 1/1600 1/200 Lytron #615 RF-2146-1.5 mg./ml. Gamma Globulin Squibb #2015. Glycine Saline Buffer p H 9.6, p = 0.24. Total Volume = 10 ml. centrations of gamma globulin 0.6 mg./ml., 1.5 mg./ml., 4.5 mg./ml., were added to each batch and tubes were processed according to the general method described. Only two suspensions of latex particles were stable when uncoited and yielded reprodncible titers when coated. These particles were Lytron-601 and Lytron-615 diameter 2500 A, and these particles were selected for further testing. Efect of p H . Detailed investigations of p H and ionic strength of the 0.25, Lytron particles have been previously presented.4,5 Stability of human gamma globulin coated latex particles was studied at pH’s varying from p H 3 to p H 11, ionic strength 0.24. The particles were stable only below p H 5.0 and above p H 8.0. Glycine saline buffer, p H 9.6 was utilized for the test procedure since there was slight instability of the coated particles at p H 8.2. Concmtrution of Added Gammu Globulin. Varying amounts of gamma globulin (Squibb #2015) were added to Lytron 615 Iatex particles (concentration 1.5 mg./mI.) a t a constant volume of 10 ml. (table 1). The concentration of added gamma globulin varied from 0.0187 mg./ml. to 15 mg./ml. The ratio of added gamma globulin varied from 5.5 x 10-6 mg./cm.z to 4.3 x 10-3 mgJcm.2 of particles. The solutions were standardized to match a 5 per cent light transmission in a Coleman Jr. Spectrophotometer. Each solution was tested with a standard rheumatoid arthritis serum and with a control of glycine-saline buffer p H 9.6. Table 1 demonstrates that coating with small amounts of gamma globulin (0.0187 mg./ml. to 0.075 mg./ml.) results in an unstable solution in both controls and test tubes. With a higher concentration of gamma globulin the particles were stable and yielded increasing agglutinating titers up to a concentration of 1.5 mg,/ml. Above this concentration (which may represent the saturation point of the particles for gamma globulin) there is no increase in titer. For this reason, the concentration of Squibb #2015 gamma globulin used to coat Lytron #615 latex particles (2500 A in diameter, 1.5 mg./ml.) in glycinesaline buffer yH 9.6 ionic strength 0.1, 25 C., was 1.5 mg./ml. brought to a total volume of 10 ml. Increase of Stability by the Addition of Albumin. Since some of the gamma globulin coated latex suspensions, even when prepared under the described optimal conditions, tended to become unstable after standing (even under refrigeration) for a period of time, albumin was added to the stock suspension as a stabilizing agent. Y Albumin 5 pg. N/ml. of stock solution was utilized to stabilize the solution since this amount did DETECTION OF XNTI-GLOBULIN FACTORS 197 not interfere with the titer or strength of agglutination. The suspension with stabilizing albumin incorporated has proved to be stable under refrigeration for at least one year. Detailed Final Method Before preparing a large stock solution of Lytron gamma globulin suspension, the optimal amount of gamma globulin to be added must be determined. One 1171. of a suspension of Lytron particles #615 (serial RF-2146) containing 48 to 50 per cent solids is diluted to 1.5 per cent of solids with distilled water. A 3 per cent solution of FII gamma globulin in glycine-saline buffer p H 9.6 is prepared. A preliminary titration is then performed. One ml. of Lytron containing 15 mg. of solid particles is placed in each of five tubes and 7 ml. of glycine-saline buffer is added to cach tube. Two ml. of gamma globulin containing 0.75 mg., 1.5 mg., 6 mg., 15 mg., or 45 mg., are added to the Lytron buffer mixture respectively. Tubes are carefully shaken to assure mixing and incubated for one hour at room temperature. The tubes are then centrifuged under refrigeration a t 4 C. for an additional hour at 12,000 r.p.m. Supernatants are discarded and the sediments washed twice with 10 ml. of glycine-saline buffer. The sediments are carefully resuspended by stirring the particles with an applicator stick. Following the second washing the sediment is resuspended in 1 ml. of glycine buffer. Of this stock solution 0.1 to 0.5 ml. is added to 10 ml. of glycine buffer until a 5 per cent transmission is reached at 650 rnp in a Coleman Jr. Model 11, Colorimeter in a 13 x 100 nim. square cuvette. The five working solutions are then added to five rows of a serially diluted rheumatoid serum (1: 10 to 1:10,240) and a control of glycine buffer. Latex gamma globulin 0.5 ml. is added to 0.5 ml. of serially diluted serum. The tubes are then incubated for 1 hour in a water bath at 56 C., refrigerated overnight at 4 C. and centrifuged the next day at 3,0008 r.p.m. for 5 minutes. The resulting visible agglutination (1+ to 4 + ) is read with the naked eye. The most suitable coated particles are those which show no agglutination in the control tubes and produce the highest agglutination titer with the standard serum. The least amount of gamma globulin consistent with the maximum agglutination titer should be selected. Once this preliminary selection of the proper amount of gamma globulin to be added to the Lytron suspension has been made, a large batch of particles may be prepared using the same proportions of material. The only differences in preparation of this large batch are that following the second washing, a glycine-saline buffer p H 9.6 is used which contains merthiolate ( 1:10,000). Also, 5 y pg. nitrogen of albumin per ml. of stock solution is addcd to preserve stability under refrigeration for at least twelve months. The stock solution is diluted as previously described to make the working solution. The test itself is performed by a technique similar to that of the standard latex test.2 Serum should not be inactivated. The wosrking pre-coated gamma globulin latex suspension is prepxed so that a 5 per cent light transmission is reached at 650 mp in a Coleman Jr., Model 11 Colorimeter in a 13 x 100 nun. square cuvette. One half nil. of this solution is added to 0.5 ml. of the serially diluted serum to be tested, utilizing glycine-saline buffer at pH 9.6. The tubes are then incubated, refrigerated and centrifuged as described in the preliminary titration and the agglutination read with the naked eye. Any titer obtained using this precoated particle system is considered positive. RESULTS The sera of 128 patients with rheumatoid arthritis, 343 with other arthritic and non-arthritic diseases and 250 controls were tested by both the standard FII LP and modified pre-coated FII LP tests. Results of the two tests closely paralleled each other, but the pre-coated FII LP yielded titers which were one 198 SINGER, PLOTZ, GOLDBERG Table 2.-Results of Precoated Gamma Globulin Latex Test in Sera with Rheumatoid Arthritis and Other Conditions Number Rheumatoid arthritis a. Adult b. Juvenile Systemic lupus erythematosus Scleroderma Osteoarthritis Gout Rheumatic fever Cirrhosis of the liver Syphilis Neoplastic disease Controls a. Apparently healthy Hospital population b. Healthy individuals of Patients Per Cent Total Total Sera Positive 120 15 30 10 120 20 45 20 40 43 82 3 9 2 2 1 2 68.3 20.0 30.0 20.0 1.8 5.0 0.8 25.0 10.0 4.6 125 2 1.6 125 1 0.8 4 5 4 to two dilutions lower. Table 2 shows the results using the pre-coated FII LP test. 68.3 per cent of adult rheumatoid arthritis patients were positive as contrasted with only 20 per cent of the juvenile rheumatoid arthritis patients. Thirty patients with systemic lupus erythematosus yielded 30 per cent positive results, a figure in agreement with that generally accepted for the standard latex test. Twenty per cent of sera from patients with scleroderma were positive but sera from patients wih rheumatic fever, osteoarthritis and gout gave, as expected, a very low incidence of positive results. An incidence of 25 per cent positive tests for anti-gamma globulin factors in sera from chronic liver disease was encountered. Sera from four of forty patients with syphilis and positive Wassermann reactions had positive titers. Only 0.8 per cent of a group of sera from 125 apparently healthy non-hospitalized individuals was positive. 1.6 per cent of an apparently healthy population (physicians, nurses, technicians) were positive, a figure in agreement with those results reported utilizing standard technics. Twenty sera from patients with rheumatoid arthritis and a positive titer of 1:2560 were tested monthly for a period of six to twelve months utilizing the stock suspension of pre-coated particles. There was no more than one tube variation during this time. DISCUSSION The FII-LP test has been widely m0dified.l Certain modifications, however, carry with them fundamental changes which may lead to great variability in results. The standard latex test utilizes only particles 0.8 micron in diameter.2 Different batches of alcohol-fractionated gamma gIobulin vary in degree of- denaturation, amount of aggregation, stability at an alkaline pH and degree of contamination by other plasma protein^.^,^ Gamma globulin prepared by sodium or ammonium sulfate fractionation may react differently due to the presence of other plasma proteins which act as stabilizers of the DETECTION OF ANTI-GLOBULIN FACTORS I 99 colloidal latex gamma globulin suspension. Pure 7s gamma globulin used for coating of the particles will be more sensitive, but less stable, than aggregated gamma g l o b u h 6 For these reasons, the present test was developed in an effort to eliminate many of the factors which lead to variability in the reporting of result^.^^^ The stability of 0.25 micron diameter latex particles (Lytron) at various pH and ionic strengths has previously been r e p ~ r t e d . The ~ , ~ amount of gamma globulin used to coat the particles was determined as described.1° If 15 mg. of 2300 A Lytron particles (2.3 x particles with a total surface of 4.1 square feet are mixed with 15 mg. of gamma globulin in a total volume of 10 ml. then interpolation in an experimentally established bonding isotherm shows that one is well within the area where the isotherm curve tends to flatten out out horizontally, that is, within the area where the amount of bound gamma globulin approaches saturation. This proportion and concentration were chosen SQ that even a decrease of up to 50 per cent in gamma globulin concentration will not appreciably diminish the amount of gamma globulin bound per unit surface of particle. The use of concentration of gamma globulin solution 10 per cent or more dilute than the recommended standard may lead to instability of the resultant latex gamma globulin suspension. In order to increase the stability of the Lytron gamma globulin suspension so that a standard batch may be kept for a year or more, small amounts of albumin ( 5 Y g.nitrogedml.) were added because of its protective capacity in colloidal suspensions. This albumin had no effect on the tests for anti-gamma globulin factors. The presently described test, using the stated precautions, is much less variable than the standard FII LP test. The technic is somewhat more difficult and this suggests that the standard latex fixation test should still be used for routine screening purposes. The new test may be performed in positive and doubtful cases and in epidemiologic and other research. One of the purposes of introduction of the new test is to produce titers which not only are reproducible from one laboratory to another but which may provide information concerning variation in titer of rheumatoid factor in the same patient at various stages of disease. Inherent in serologic testing is a small titer variation which in general should not exceed two dilution tubes. Various rheumatoid sera were tested by the new technic using a single batch of pre-coated particles over a period of 1 ear.^,^ Only one tube dilution variation was noted. The new test is therefore well within the normal limits of variability. Reproducible titers are important in studies which reflect possible changes in rheumatoid factor according to the stage and seventy of disease, to determine whether there is a change in reactivity of patient serum with various methods of treatment, to determine whether there is a relationship between titer and prognosis and to reflect possible increases in titer during such complications of rheumatoid arthritis as vasculitis. Epidemiologic studies, both of a static and progressive nature, will benefit by the use of a test which yields reproducible titers. Since the presence of any anti-gamma globulin factors in serum is abnormal, any positive titer utilizing 200 SINGER, PLOTZ, COLDBERG the relatively stable technic here described should be considered as a positive test. The present test utilizes pre-coated Lytron which has so far proved to be stable under refrigeration for at least one year. It may possibly prove to be stable much longer. Laboratories may thus prepare their suspensions at less frequent intervals and some central agency might prepare the pre-coated particles and distribute them to various laboratories, further reducing the possibility of variability of results due to changes in technic. In studies of the presence of anti-gamma globulin factors, both in rheumatoid arthritis and in other diseases, standardized results are essential. This is true both of the present te3t and of the usual latex fixation and sensitized sheep cell test. Particular care should be used in the selection and preparation of particles, gamma globulin and buffer, and they should be tested against standard rheumatoid sera which also should be made available by some central agency. A kit might be provided containing all of the materials for the test, to serve as reference for the materials utilized by each laboratory. It has been demonstrated that when an animal is immunized with denatured homologous gamma globulin the resultant antiserum is reactive with the -gamma globulin of other species.11-13In the FII-LP test adsorption of 7s human gamma globulin on the surface of the latex particles produces denaturation or at least distortion of the native human gamma globulin molecules. This surface denaturation uncovers reactive sites for human antibodies to rabbit and other animal gamma globulins. The sensitized sheep cell test, on the other hand, detects antibodies to rabbit gamma globulin. Various studies have shown that anti-gamma globulin factors exhibit 3 serologic patterns of reactivity with human and rabbit gamma g l o b ~ l i n . l These ~ - ~ ~ patterns are: (1) reactive with human gamma globulin; ( 2 ) reactive with rabbit gamma globulin; ( 3 ) reactive with both human and rabbit gamma globulin. The great majority of rheumatoid sera react with both human and rabbit gamma globulin. However, occasional sera are reactive only with rabbit gamma globulin in the SSC test. Sera from patients with acute and chronic inflammatory states not related to connective tissue disease frequently follow the pattern of being reactive only with human gamma globulin, i.e. positive FII LP and negative SSC. However, some sera from this disease group are reactive with both FII LP and SSC, and very few with SSC only. It is also known that in this group of patients FII LP titers tend to fall as the patient imp r o v e ~For . ~ this ~ ~reason ~ ~ the development of a test which yields reproducible results may result in the accumulation of more consistent and therefore more reliable data. SUMMARY AND CONCLUSIONS 1. A modified latex agglutination reaction utilizing pre-coated latex particles 2300 A t 200 A in diameter has been described. Optimal conditions for coating particles with gamma globulin are delineated. 2 . This modification yields reproducible titers and eliminates doubtful results. 201 DETECTION OF ANTI-GLOBULIN FACTORS 3. The latex-gamma globulin suspension is stable under refrigeration for at least 1year. REFERENCES I. Singer, J . M.: The latex fixation test in rheuniatoid clisease. Am. J. Med. 31: 766, 1961. 2. -, Plotz, C.: The latex fixation test. I. Application to the serologic diagncsis of rheumatoid arthritis. Am. J. Med. 21:888, 1956. 3. Hozsoky. S.: The problem of standardization in rheumatoid arthritis serology. Arth. & Rheumat. 6:641, 1963. 4. Halbcrstam, D., Singer, J. M., Allen, E. G. and Plotz, C. M.: Stability characteristics of 0.2 micron diameter polystyrene latex particles. Atti. del X Congresro dela lega Internazionale contro il rheumatismo., 2:854, 1961 hfinerva hfedica. .5. Oreskes, I., and Singer, J. M.: Influence of pH, ionic strength and protein concentration on stability of polystyrene latex siispensions. Proc. Soc. Biol. & Med. 115:753, 1964. 6. Singer, J. M., Altmann, G., Goldenberg, A. and Plotz, C. M.: The mechanism of particulate carrier reaction with rheumatoid sera. 11. Sensitizing capacity of various human gamma globulin for latex particles. Arth. & Rheumat. 3:515, 1960. 7. -, -, Oreskes, I. and -: The mechanism of particulate carrier reaction 111. The stabilizing effect of serum protein. Am. J. Med. 30:772, 1961. 8. -, Plotz, C. M. and Eason, E.: The latex fixation test IV. The serological app1ic:ition of 0.2 micron diameter latex particles coated with gamma globulin. Atti. del X Congress0 della lega Internazionale contro il Rheumatismo. 1:304, 1961. 9. -, - and Goldberg, R.: A technique of prccoating polystyrene lytron particles with gamma globulin for the detection of macroglobulins which react with human gamma globulin. Third Pan-American Congress on Rheumatic Disease Santiago, Chile, October, 1963. 10. -, Oreskcs, J., Hutterer, F. and Emst, J.: The mechanism of particulate carrier rractions V. Adsorption of human gamma globulin to 0.2 micron diameter latex particles and their agglutination by rheumatoid factor. Ann. Rheum. Dis. 22:434, 1963. 11. Milgrom, F., and Witebsky, E.: Stndies on the rheumatoid and related serum factors. J. A. M. A. 174:56, 1960. 12. Abnlzzo, J. L., and Christian, C. L.: Induction of rheumatoid factor-like substance in rabbits. Arth. & Rheumat. 4:103, 1961. 13, klcCluskey, T. R,, Miller, F. and Benacerraf, B.: Sensitization to denatured autologous gamma globulin. J. Exper. Med. 115:253, 1962. 14. Vaughan, J. H.: Behavior of the rheumatoid arthritis agglutinating factor with immune precipitates. J. Immun01. 77:181, 1956. 1.5. LoSpalluto, J. and Ziff, M.: Chromatographic studics of the rheumatoid factor. J. Exper. Med. 110:169, 1959. 16. Heimer, R., Schwartz, E. R. and FrVberg, R. H.: Different rheumatoid factors in the serum of one patient with rheumatoid arthritis. J. Lab. & Clin. Med. 57:16, 1962. 17. Milgrom, F., Witebsky, E., Goldstein, R. and Loza, U.: Studies on the rheumatoid and related serum factors. 11. Relation of anti-human and anti- rabbit gamma globulin factors in rheumatoid arthritis serums. J. A. M. A. 181:106, 1962. 18. Williams, R. C., Jr., and Kunkel, H. G.: Separation of rheumatoid factors of different specificities using columns conjugated with gamma globulin. Arth. & Rheumat. 6:665, 1963. 19. Dressner, E . and Trombly, P.: The latex fixation reaction in non-rheumatic disease. New England J. Med. 261:981, 1959. 20. Williams, R. C., and Kunkel, H. G.: Rheumatoid factor, complement, and conglutinin aberrations in patients with subacute bacterial endocarditis. J. Clin. Invest. 41:666, 1962. 202 SINGER, PLOTZ, GOLDBERG Jacques M . Singer, M.D., Head, Department of Microbiology, Division of Laboratories, Montefiore Hospital, Bronx, N e w York; Clinical Associate Professor of Medicine, State University of N e w York, Downstate Medical Center, Brooklyn, N e w York. Charles hl. Plotz, M.D., LMed. Sc.D., Clinical Associate Profesof Medicine, Director Training Program in Rheumatic Diseuse. State University of N e w York, Downstate Medical Center, Brooklyn, N e w York. SOT Rosalie Coldberg, B.A., Research Assistant, Department of Microbiology, Division of Laboyatories, Montefiore Hospital, Bronx, N e w York.