Japan. J. Med. Sci. Biol.,31,105-118,1978

SPECIFICITY OF ANTI-HUMAN IgG 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 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 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 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 : 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. Among the precipitating antisera, only anti-human IgG serum showed a high titer (1:4096), while anti-C3 and anti-C4 did not agglutinate anti-D-coated red cells. No agglutination of anti-D-coated

TABLE I Anti-human IgG activities of antiglobulin sera 1978ANTI-IgG IN ANTIGLOBULIN SERA109

red cells was observed with Rf under our experimental conditions. Human IgG-coated latex reagent was agglutinated by all the sera contain- ing anti-IgG antibody in contrast to anti-D-coated red cells which remained unagglutinated by Rf. Moreover, it was unnecessary to exclude possible in- volvement of anti-erythrocyte antibodies in antiglobulin sera as in the case of anti-D-coated red cell agglutination. However, in Coombs sera, titers against human IgG-coated latex were found much lower than those of anti-D-coated red cell agglutination by the same sera. In this respect, reaction pattern of Coombs sera was in contrast to Rf which was positive in human IgG-coated latex agglutination up to 1:128 but was negative in anti-D-coated red cell agglutination.

Anti-human IgG Antibody Response to Immunization with Freund's Adjuvant Since most of the Coombs sera except FH were thought to have been prepared by immunization using Freund's adjuvant, we tried to follow the course of anti-human IgG antibody response in rabbits immunized by the method, especially in early stages of immunization (Table II). In 7 days after the first immunization, anti-D-coated red cell agglutination was already positive

TABLE II Appearance of anti-human IgG antibody by immunization with Freund's complete adjuvant (earlier stages of immunization) 110ARAI et YASUDAVol.31

TABLE III

Appearance of antibody activity and its localization in immunoglobulin

fractions in rabbits immunized by the method according to Haynes •¬ Chaplin

* n .t.: not tested with titers of 1:4-1:64; and its titer elevated to 1:32-1:4,096 in the following 9 days. On the other hand, human IgG-coated latex agglutination was negative in all the five rabbits on the 7th day after the first immunization and at best 1:2 on the 16th day. Thus, it was found that by this most commonly used method of immuni- zation, anti-human IgG activity detectable by IgG-coated latex reagent was very weak even in early stages of immunization.

Anti-human IgG Antibody Response Following Haynes and Chaplin's Method Next, we tried to follow the time course of anti-human IgG antibody response and the localization of the activity in immunoglobulin classes in rabbits intraperitoneally immunized by the method of Haynes and Chaplin (Table III). In rabbit C-003, the ratio of the agglutination titer of anti-D-coated red cells to that of human IgG-coated latex was 4/1 on the 7th and 10th days, but the value grew greater in later stages of immunization. The agglutination titer of anti-D-coated red cells elevated until the 15th day in IgG fraction, but fell in IgM fraction after reaching its peak on the 10th day. Antibody activity against human IgG-coated latex was always present in both of IgG and IgM fractions. 1978ANTI-IgG IN ANTIGLOB ULIN SERA111

TABLE IV Appearance of antibody activity in antisera against aggregated human IgG

In C-004, most of the activity against human IgG-coated latex was found to be localized in IgM fraction. In C-006, in which antibody response was generally weak, activity against anti-D-coated red cells was localized in IgG fraction alone. In this rabbit, activity against human IgG-coated latex was already present in serum before the first immunization and it was located in IgM fraction. In 10-14 days of immunization, the activity appeared in IgG fraction, too, but soon disappeared. On the 24th day, activities against anti-D-coated red cells and human IgG-coated latex was found to be separately localized in IgG and IgM fractions, respectively. A similar tendency was observed in rabbit C-004, 23 days after the first injection. From these results, it can be seen that IgG-latex test usually became positive when considerable response of IgM antibody was recognized.

Immunization with Aggregated Human IgG By immunization with SDS-aggregated IgG, anti-D-coated red cell aggluti- nation remained negative or very weak until the 29th day, despite the rise in the titer of IgG-coated latex agglutination. Thus, the reaction pattern of these antisera was similar to that of Rf, although the titer of human IgG-coated latex agglutination test was lower than that by Rf. In anti-heat-aggregated IgG serum, reaction pattern was similar to that of HC (Table IV). 112ARAI et YASUDAVol.31

TABLE V Absorption of antiglobulin sera with human IgG-coated latex particles

Numerals indicate the minimum amount (mg) of human IgG samples required for the inhibition of agglutination by appropriately diluted antisera. * n.t.: not tested, because the reaction was negative or too weak.

Specificity o f Anti-human IgG Antibody

Antiglobulin sera (FGA, HC and FH) and aH were absorbed with latex particles coated with heat-aggregated human IgG. As shown in Table V, aggluti- nation titer against anti-D-coated red cells decreased from 1:32-1:256 to less than 1:2. By absorption with uncoated latex particles there was no decrease in titer observed. Thus, the anti-IgG antibodies detected by anti-D-coated red cells must have been removed by its binding to human IgG-coated latex particles. In the inhibition experiment with human IgG (Table VI), however, aggluti- nation of anti-D-coated red cells by commercial Coombs serum and by FGA seems to have been better inhibited by native IgG(N) than by aggregated one (A). Antiglobulin activity of other sera always appeared to have been better inhibited by aggregated human IgG(A). For example, with Rf which is generally thought to be better inhibited by A than by N, the ratio N/A was a little higher than 1978ANTI-IgG IN ANTIGLOBULIN SERA113

Fig. 1. Immunodiffusion with various antisera against aggregated human IgG. HC: Coombs serum prepared by the method of Haynes and Chaplin (C-003;10 days; A: SDS-aggregated human IgG Rf: rheumatoid arthritis serum aS: anti-SDS-aggregated IgG serum (aS-1) FH: Coombs serum prepared by the method by Fairley & Harris G: anti-y chain serum aH: anti-heat-aggregated IgG serum

10/1. The sample N was found by ultracentrifugal analysis to be almost ex- clusively consisted of 7S component, while A of 53S ones. Our antisera except commercial Coombs serum and FCA formed precipi- tation bands on agar plate against SDS-aggregated human IgG, as did Rf. None of them formed a precipitation band against native human IgG. With some of HC, a precipitation band was also formed against light chains of human immunoglobulin. Precipitation bands formed by FH and HC fused with those by aH, aS, Rf and anti-r-chain serum (Fig. 1). When aggregated human IgG was added to aS or HC, the precipitation band was no longer formed against aggregated IgG (Fig. 2). On the other hand, addition of native human IgG did not affect precipitation with these sera against aggregated human IgG. From these results, anti-IgG antibody in HC, FH and aS seems to be different in specificity from antibody in FCA or in commercial Coombs serum, in that the specificity of the former is directed to more aggregated IgG molecules.

Species Specificity of Anti-IgG Antibodies Table VII shows the results with some of antiglobulin sera in Waaler- Rose test employing sheep red cells sensitized with IgG fraction obtained from commercial anti-sheep haemolysin of rabbit origin. Some of FH and HC as well as aS were found to have agglutinated sensitized sheep red cells to the dilution of 1:16 to 1:256. The activity was localized in 114ARAI et YASUDAVol.31

Fig.2. Inhibition of immunodiffusion by treatment with aggregated IgG. A, aS, HC: same as in Fig.1 +N: treated with •gnative•h human IgG +A: treated with aggregated human IgG 20ƒÊl of samples of human IgG (N: 0.3 mg; A: 0.28 mg, respectively) was mixed with undiluted antisera.

TABLE VII Anti-rabbit IgG activities in various antiglobulin sera

IgM fraction of the immune sera. When latex particles coated with heat- aggregated rabbit IgG were used, no agglutination was observed with any of the sera. Only weak reaction was observed with IgM fractions separated from some of HC. When the sera of aS, FH and HC were absorbed with latex particles coated with aggregated rabbit IgG, only Waaler-Rose test became negative without affecting titers in anti-D-coated red cell agglutination. Conversely, in all the sera, except C-006 (10 days), absorption with human IgG-coated latex removed antiglobulin activity to anti-D-coated red cells alone, with no or slight decrease in titer in Waaler-Rose test. Thus, it was found that in these sera, anti-human and anti-rabbit IgG factors were co-existent independently to each other. 1978ANTI-IgG IN ANTIGLOBULIN SERA115

TABLE VIII Absorption of C-006 (10 days)

Only in C-006, absorption with human IgG-coated latex removed activity to IgG of both of the species (Table VIII). So, in this serum, a factor capable of cross-reacting to IgG of both of the species was demonstrated.

DISCUSSION

In serological characterization of antiglobulin factors, conventional Coombs serum can be placed in one extreme opposite to the other end occupied by Rf. Unique feature in its reaction pattern is that, in contrast to Rf, anti-D-coated red cells are agglutinated to high titers whereas very low titers were observed with human IgG-coated latex reagent. Since the activity of Rf is known to be mostly localized in IgM, it was thought as if human IgG-coated latex agglutination could be strongly positive with Coombs sera at earlier stages of immunization, in which it is likely to be able to detect antibody response in IgM fraction. However, even in a very early stage of immunization with Freund's complete adjuvant, human IgG- coated latex agglutination was found at best weakly positive despite considerable rise in titer in anti-D-coated red cell agglutination. On the other hand, by following the immunization method of Haynes and Chaplin (1971), anti-IgG response which could be reflected to human IgG-coated latex agglutination was readily observed. As reported by Haynes and Chaplin (1971), there was a peak in activity in IgM fraction against anti-D-coated red cells which appeared 10-15 days after the first immunization. Then, its antibody activity became gradually localized mainly in IgG fraction. The reactivity against human IgG-coated latex was found in IgM and IgG fractions, but be- came more or less confined to the former. In general, human IgG-coated latex agglutination test became positive in antiglobulin sera obtained by such immu- nization methods capable of eliciting considerable IgM antibody response, although the activity was not always confined to IgM fraction. Specificity of anti-human IgG antibodies which can be detected with each of the two reagents was then studied. From the results of absorption experiments with human IgG-coated latex particles, the antibodies detected with each of the reagents were found to be closely related in specificity. 116ARAI et YASUDAVol.31

When they were studied by agglutination inhibition tests, however, anti-IgG antibody activity in antiglobulin sera other than FCA and commercial Coombs serum appeared to have been better inhibited by aggregated, rather than by •g native•h, human IgG. Also in the immunodiffusion test, HC and FH antisera formed a precipi- tation band against aggregated human IgG, as did Rf. The precipitation band fused with those formed by aS, Rf and anti-ƒÁ-chain serum, and can be regarded consequently as being formed against antigenic determinants in ƒÁ-chain of aggregated human IgG molecules. Although aS was prepared by intravenous immunization while HC sera by intraperitoneal immunization, it is likely that similar antigenic modification might have occurred in IgG molecules which are contained in both of the immunogens. In some of our antiglobulin sera, we observed agglutinating activity against

sheep red cells sensitized with rabbit IgG. In most of them, anti-human and anti-rabbit IgG factors could be separately removed through absorption with latex particles coated with human and rabbit IgG, respectively. In only one of them, antiglobulin factor cross-reacting to IgG of both of the species could have been demonstrated. Anti-rabbit IgG activity in our antiglobulin sera was localized in IgM fraction of the sera of immunized rabbits and in this respect, it must be some- thing other than 7S •ghomoreactant•h against determinant(s) which would be

Fig.3. AntiseraReaction : patternsFCA Antiglobulin ofHC} various anti-human(Coombs) seraIgG factors

aS : antisera against SDS-aggregated IgG Rf : rheumatoid arthritis sera *A: aggregated human IgG *N: native 1978ANTI-IgG IN ANTIGLOB ULIN SERA117

exposed by enzymatic treatment (Mandy, Fudenberg and Lewis,1965). There has been a report by Milgrom and Witebsky (1960) that anti-human IgG antibody was produced in rabbits when immunized with rabbit IgG. Such broader spectrum of species-specificity is considered to be characteristic in •gauto-reactive•h antiglobulin factors. Even in sera of normal rabbits, anti-human IgG factor which is detectable by human IgG-coated latex reagent was demonstrated

(Watson and Collins,1963; Yasuda and Kano,1976). Specificity of various antiglobulin sera is diagramatically summarized in Fig.3.

Specificity of our Coombs sera and anti-human aggregated IgG sera can be shown as locating between the two extremes: Rf on the one end and FCA on

the other, and being directed toward antigenic determinants formed or exposed in more or less aggregated human IgG molecules. Agglutination of latex parti- cles coated with human IgG seems to be a better indicator for the antibodies to aggregated human IgG than anti-D-coated red cell agglutination. According to Singer et al. (1960), IgG-coated latex agglutination is different in specificity from that of agglutination of tanned red cells coated with human IgG which is sometimes used in evaluation of Coombs sera (Engelfriet et al.,1968). Therefore, the two systems can not substitute for each other in studying specificity of anti- human IgG antibodies. In clinical administration of human IgG to patients, especially of prepara- tions for intravenous use, more attention has been paid to the acquisition of antigenicity to humans as a result of aggregation of IgG molecules. In this report, it has been shown that Coombs sera prepared by various methods of

immunization may show difference among themselves in their specificity to native or aggregated human IgG, and thereby they may be used for differentiat- ing antigenic modifications in human IgG molecules caused by aggregation, together with other antiglobulin factors such as rheumatoid factor.

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