Japan. J. Med. Sci. Biol.,31,105-118,1978 SPECIFICITY OF ANTI-HUMAN IgG ANTIBODIES IN ANTIGLOBULIN (COOMBS) SERA KAYOKOARAI and JUNICHIYASUDA Central Laboratoryfor ClinicalInvestigation, Osaka UniversityHospital, Fukushima-ku,Osaka 553, and Departmentof GeneralBiologics Control, National Institute of Health, Shinagawa-ku,Tokyo 141, Japan (Received:August 22,1977) SUMMARY: The agglutination test with latex particles coated with aggre- gated human IgG was introduced into the evaluation of Coombs serum as an additional test for anti-IgG antibody activity. In Coombs sera prepared by the conventional immunization method employing Freund's adjuvant, latex agglutina- tion titers were found much lower than those of anti-D-coated red cell agglutination. On the other hand, in sera prepared by other immunization methods, such as the one according to Haynes and Chaplin (1971), anti-IgG antibody response was readily observed by IgG-coated latex agglutination. Specificity of anti-IgG antibodies in the latter sera seems to be predominantly directed to aggregated human IgG. INTRODUCTION In evaluation of anti-human globulin (Coombs) serum for its antibody spectrum and potency, human red cells coated with various blood group alloanti- bodies are used for testing anti-human IgG antibody activity thereof (ICSH/ISBT Expert Panel, Working Party,1973). In this respect, Coombs serum can be regarded as an anti-human IgG factor produced in heterologous species by deliberate stimulation. Here, attention was paid to the species-specificity of anti-IgG antibody rather than to the capability of differentiating the antigenic modifications within human IgG molecules caused by aggregation or denatu- ration. On the other hand, antigenic modifications in human IgG molecules have been studied by means of their reactivity to antiglobulin factors such as rheumatoid factor. For example, denaturation of human IgG molecules by physicochemical treatments could have been characterized by its acquisition of ability of forming a precipitation band against rheumatoid factor (Edelman, Kunkel and Franklin,1958; Henney and Ishizaka,1968). Agglutination of latex particles coated with heat-aggregated human IgG is widely used for detect- ing rheumatoid factor (Singer and Plotz, 1956), together with Waaler-Rose test which employs sheep red cells sensitized with rabbit antibody (Rose et al.,1948). 新井加余子(大 阪大学医学部附属病院 中央臨床検査部 大阪市福島区堂島浜通3-1/2) 安田純一(国 立予防衛生研究所 一般検定部) 105 106ARAI et YASUDAVol.31 The present investigation aimed at introducing human IgG-coated latex reagent from serology of rheumatoid factor into the evaluation of Coombs sera and thereby characterizing anti-human IgG antibodies from the viewpoint of reactivity to native or aggregated human IgG molecules. MATERIALS AND METHODS Antiglobulin (Coombs) sera: 1) Commercial Coombs sera: From commer- cially purchased Coombs sera listed in Table I, anti-human serum (rabbit) No.7718-1 from Ortho Diagnostics (Raritan, NJ, U.S.A.) was selected to be used in subsequent experiments. 2) Antisera prepared by immunization with Freund's complete adjuvant (hereafter be abbreviated as FCA): Ten milligrams of human IgG mixed with FCA (Difco, No.0638-60) was injected into foot pads of rabbits. Immunization schedule was modified from the one recommended by Ironside (1968). 3) Antisera obtained by the immunization method of Fairley and Harris (1962) (hereafter be abbreviated as FH); One milliliter of washed rabbit red cell sediment was incubated with 10 ml of human IgG (10 mg/ml) at 37 C for an hour. After washed three times, the red cells were suspended in physiological saline up to 3 ml. Each 3 ml of thus prepared red cell suspension was intra- venously injected into rabbits twice a week and blood was collected 7 days after the fourth injection. 4) Antisera obtained by the immunization method of Haynes and Chaplin (1971) (hereafter be abbreviated as HC): Rabbits were intraperitoneally immu- nized with human IgG adsorbed to aluminium hydroxide gel. Human IgG: Human Normal Immunoglobulin, No.266 was obtained from the Green Cross Corporation, Osaka, Japan. Its IgG content determined by free-boundary electrophoretic analysis was 98.9%. •g Native•h human IgG: Sodium sulfate was added to a human IgG solution (20 mg/ml) to a final concentration of 0.62 M according to the method by Christian (1958). Its centrifuged (1000•~g,3 hr) supernatant solution was dialyzed against buffered saline (pH 8.0 by mixing 0.15 M Na2HPO4 and 0.15 M KH2PO4 and diluted with an equal volume of physiological saline). •g Heat-aggregated•h human IgG: A human IgG solution (10 mg/ml) was heated at 63 C for 20 min according to Henney and Ishizaka (1968). Immediately after cooled, sodium sulfate was added to 0.36 M. The resulting precipitate was dissolved in buffered saline (pH 8.0) described above and dialyzed against the same buffered saline solution. •g SDS-aggregated•h human IgG: Human IgG (20 mg/ml) was mixed with an equal volume of 0.05 M sodium dodecylsulf ate solution according to Henney and Stanworth (1965) and kept at 37 C for 2 hr. Then, it was dialyzed for 2 days in the cold against 0.2 M phosphate buffer (pH 7.5). Its centrifuged (2,000 rpm, for 3 hr) sediment was dissolved. Antisera against aggregated human IgG: Heat-aggregated or SDS-aggregated 1978ANTI-IgG IN ANTIGLOBULIN SERA107 human IgG was intravenously injected into rabbits , each 5 mg per injection (Biro and Garcia, 1965). Infection was given weekly and blood was collected 5 days after the third injection (hereafter be abbreviated as aH and aS , respec- tively). Rabbit IgG: IgG fraction isolated by chromatography on Sephadex G-200 from commercial rabbit anti-sheep erythrocyte serum (Nankai Kagaku No .827) was concentrated. Heat-aggregated rabbit IgG: Rabbit IgG was treated by the same method as described in •gheat-aggregated human IgG•h . Other commercial reagents: Precipitating anti-human IgG serum (Goat , N o.3) was obtained from Research Foundation for Microbial Diseases of Osaka University, Osaka, Japan. Precipitating anti-human complement sera , anti-h uman C3 (A 432) and anti-human C4 (A 4333) , were supplied by Meloy La- boratories, Inc. (Springfield, IL, U.S.A.). Human Bence Jones proteins type K (672 AA) and type L (772 AA) were purchased from Behringwerke, Marburg/ Lahn, Germany. Anti-D (Rho) blood typing serum (albumin agglutinating serum) R-5542-2 was purchased from Ortho Diagnostics Inc . (Raritan, NJ, U.S.A.). Rheumatoid arthritis serum (abbreviated as Rf): Fifty sera from patients with clinical diagnosis of rheumatoid arthritis giving positive results in Waaler - Rose and FII latex fixation tests at the Central Laboratory for Clinical Investi- gation, Osaka University Hospital were pooled. Fractionation o f sera: IgG and IgM fractions were separated by molecular sieve chromatography through a Sephadex G-200 column , followed by concen- tration to the original volume of serum (Porath , 1960). Coating of latex particles with aggregated rabbit IgG: One volume of 5% suspension of Bacto latex particles (0 .81 p, Difco Laboratories, Detroit, MI , U .S.A.) was mixed with nine volumes of aggregated rabbit IgG (10 mg/ml) and kept for 4 hr at room temperature and thereafter overnight at 4 C . It was centrifuged at 8,000 rpm for an hour and washed twice with glycine buffer (pH 8.0) prior to resuspension in the same buffer to the original volume of the latex suspension. Latex particles coated with (aggregated) human IgG: RA test reagent (IATRON, Tokyo, Japan, No. 273) was used. Agglutination of anti-D-coated red cells: The technique specified by the Working Party of the ICSH/ISBT Expert Panel for the second practical trial (1973) was followed with slight modifications. Five per cent suspension of group O, D (Rho)-positive human red cells was incubated with 1:2 diluted anti-D serum at 37 C for an hour and then washed three times . Fifty microliters of 5% suspension of anti-D-coated red cells was mixed with the equal volume of twofold dilution series of test serum and the results were read macroscopically after centrifugation at 1,000 rpm for a minute. Physiological saline was used instead of buffered saline specified by the Working Party (1973) . Latex agglutination test: Fifty microliters of a test serum was mixed on a black plate with the equal volume of a suspension of latex particles coated 108ARAI et YASUDAVol.31 with human or rabbit IgG. The plate was gently rocked for a minute and the results were read macroscopically. Waaler-Rose test: The technique described by Heller et al. (1949) was followed with one-tenth reduction of the reaction volume. Absorption o f antiglobulin sera by latex particles coated with IgG: A 0.2-ml portion of serum to be absorbed was mixed with the equal volume of a suspension of latex particles coated with human or rabbit IgG. The mixture was left for an hour at room temperature and thereafter at 4 C overnight. The supernatant fluid was separated by centrifugation at 8,000 rpm for an hour. Inhibition of agglutination by human IgG: Fifty microliters of dilution series of human native or aggregated IgG were mixed with 50 ƒÊl of an appro- priately diluted test serum and the mixture was kept at room temperature for 20 min. Protein content of stock solutions of IgG was approximately 3.5 mg/ml when determined by the Folin-Ciocalteau method. Double immunodiffusion: Agarose (Behringwerke, No.17F 5204) and Special Agar Noble (Difco, 0142-01) were dissolved in a mixture of one volume of 0.067 M phosphate buffer (pH 7.4) and four volumes of physiological saline, each to a final concentration of 0.5%. It was poured into plates and used in the double immunodiffusion technique. RESULTS Reaction Pattern of Anti-IgG Antibody in Coombs Sera As shown in Table I, agglutination titers of Coombs sera were 1:64 to 1:1024 against anti-D coated red cells.