Clin. exp. Immunol. (1981) 46, 541-546.

A simple quantitative assay of circulating immune complexes by laser nephelometry, using a rabbit IgG antibody against human aggregated IgG

K. ARAI & A. SHIMIZU Central Laboratory for Clinical Investigation, Osaka University Medical School, Osaka, Japan

(Acceptedfor publication 12 June 1981)

SUMMARY

Circulating antigen-antibody complexes were detected by measuring agglutination of complexes with rabbit antisera against heat-aggregated human IgG by means of laser nephelometry. Rabbit antisera were obtained by immunization with heat-aggregated human IgG after intravenous injection of a large amount of native human IgG. The antisera were observed to be almost specific to conformationally altered human IgG by immunodiffusion. Even ifthe antibody activity to native IgG did occur in the antisera, the native IgG in test sera was in excess of antigen in the assay system employed for the detection ofimmune complexes. In conclusion, the minor antibody activity against native IgG does not interfere with the assay of immune complexes by laser nephelometry. The clinical applications demonstrated the advantage of the method.

INTRODUCTION Various techniques have hitherto been proposed for determining circulating immune complexes (CIC) in serum, some of which have been approved as suitable for application to clinical investigation of CIC (Lambert et al., 1978; Zubler et al., 1976; Hay, Nineham & Roitt, 1976; Sobel, Bokisch & Muller-Eberhard, 1975; Lurhuma et al., 1976; Casali et al., 1977; Luthra, McDuffie & Samayoa, 1975; Cowdery, Treadwell & Fritz, 1975; Theofilopoulos, Wilson & Dixon, 1976). These methods, however, require purification of proteins, maintenance of cell lines or reagents from patients. An attempt was made by the authors at accomplishing a simple, accurate and non-isotope assay for CIC. Injection of a large amount of antigen into animals is commonly known to inhibit the development of normal immune responses. Animals lose responsiveness to the antigen. In the experiments described herein, massive doses ofnative human IgG (NHG) were injected into rabbits which were then immunized with an appropriate amount of heat-aggregated human IgG (AHG). This procedure produced antisera specific for aggregated IgG. Agglutination of CIC with anti-AHG was measured by laser nephelometry. The native IgG in test sera did not interfere with the assay.

MATERIALS AND METHODS IgG. Human IgG (Kabi) was dissolved in phosphate-buffered saline (PBS) (0 05 M phosphate, 0- 15 M NaCl, pH 7-2) at a concentration of7 mg/ml. This solution was centrifuged at 145,000g for 1 hr and the supernatant was used as NHG. Correspondence: Kayoko Arai, The Central Laboratory for Clinical Investigation, Osaka University Hospital, 1-1-50 Fukushima, Fukushima-Ku, Osaka 553, Japan. 0009-9104/81/1200-0541$02.00 (0 1981 Blackwell Scientific Publications 54I 542 K. Arai'& A. Shimizu The method of Dickler (1974) was adopted for the preparation of AHG. Human IgG dissolved in PBS at a concentration of 7 mg/ml was heated for 20 min at 630C, cooled in an ice bath and centrifuged at 145,000 g for 1 hr and the precipitates.were used after dissolving in PBS, pH 8 0, as AHG. Protein concentration of the solution was measured by the dye-binding assay (Bio-Rad protein assay kit). Specific antisera against immune complexes. Normal adult rabbits were injected intravenously with 10 mg of NHG on day 0. Ten or 100 pg of AHG together with Freund's complete adjuvant were injected intramuscularly into rabbits on day 2 followed by reinjection of the same amount of AHG on day 16. The blood was collected on day 23. Immunodiffusion analysis. The Ouchterlony double-diffusion test was carried out in 1% agarose in 1/15 M phosphate buffer, pH 7 2 (PB). The reactions of rabbit antisera thus prepared and anti-human IgG antisera (Hoechst-Behring) to AHG (1 mg/ml), NHG (1 mg/ml) and fresh normal human serum were analysed by immunodiffusion. Standard curvesfor CIC quantitation. AHG was used as a reference for CIC quantitation. The mixture containing 500 pI of diluted anti-AHG sera ( x 30 in PB), 50 pI ofAHG solution in various concentrations (5-500 pg/ml) in PB and 450 pI ofPB containing EDTA (final concentration 10 mM) was prepared, after which it was incubated for 2 hr at room temperature. Relative light scatter (RLS) of agglutinations of AHG and antibody was determined by means of a laser nephelometer (Nephelometer PDQ, Hyland). The control solution ofAHG in PB showed a low level ofRLS. The actual RLS by agglutination was obtained by subtracting RLS ofAHG in each concentration from the total value of the reaction. AHG could be preserved in a lyophilized form without any change in its antigenicity. AHG solution in PB was divided into small tubes and lyophilized. At the time of use, it was reformed to the original volume of solution by adding deionized water. AHG dissolved in normal human serum (NHS) was also tested. Fifty microlitres of serum that contained each concentration of AHG were mixed with anti-AHG sera and PB, upon which RLS determination was made by the same method as AHG dissolved in PB. Detection ofCIC. A mixture of 50 pl oftest serum with 500 pI ofdiluted antisera and 450 PI ofPB containing 20 mm EDTA was prepared. For the control, 50 pl of test serum were added to 950 p1 of PB containing EDTA (final concentration 10 mM). The mixture was incubated for 2 hr at room temperature. The CIC values, estimated from the standard curves drawn with AHG in PB, were expressed as pg equivalents of AHG/ml. Tests were made on sera from about 300 patients with various diseases. The sera were kept at - 80'C until the time of use. Sera from clinically healthy subjects were also tested. Determination ofthe immunoglobulin class ofrabbit antibody against aggregatedhuman IgG. The rabbit antiserum against AHG prepared as described above was fractionated by gel filtration. Two millilitres of serum were applied to a Sephadex G-200 column (2-0 x 60-0 cm) and eluted with phosphate-buffered saline. Three major fractions were obtained, each peak containing mainly IgM, IgG and albumin respectively. The immunodiffusion confirmed that the IgG fraction reacted to the anti-rabbit IgG goat serum but not to the anti-rabbit IgM goat serum. The antibody solution collected from the parts of each peak giving the highest optical density was used for examining the reaction to AHG. The concentration of antibody in the solution of each peak was estimated to be approximately 1/20 that of the original serum. Without concentrating the fraction, the antibody in the gel filtration eluate reacted strongly to AHG. The reaction of fractionated antibody to AHG was tested by means of nephelometry and immunodiffusion. For the nephelometry performance, 500 pI of fractionated antibody were mixed with AHG in PB in various concentrations (5-2,000 pg/ml). After incubation at room temperature for 2 hr, the binding was measured with a nephelometer.

RESULTS Characterization ofrabbit antisera. The antisera from the rabbits immunized with AHG after massive injection of NHG reacted with IgG-coated latex (Eiken Co. Ltd, Tokyo) and erythrocytes Immune complex assay 543 sensitized with anti-D antibody (Ortho). The specificity of the antisera was examined by immunodiffusion. The antisera were seen to react to AHG as shown in Fig. 1 but failed to react either to NHG or to fresh normal human sera. In contrast, anti-human IgG sera reacted to both AHG and NHG. To arrive at the best possible procedure for obtaining the antisera specific to AHG, investigation was conducted into various combinations of antigen doses and intervals between injections, as a result ofwhich the protocol described under Materials and Methods proved to be the best ofall. The antisera from all 13 rabbits immunized so far by this procedure displayed a similar precipitin line in immunodiffusion (Fig. 1) and were observed to be specific to conformationally altered IgG. CIC quantitation. Fig. 2 shows the time variation of the reaction of AHG (100 jg/ml) and anti-AHG antisera (diluted x 30) at room temperature. The reaction began immediately after mixing and attained a plateau in 2 hr, which lasted for at least 20 hr. RLS was measured with a laser nephelometer after incubation for 2 hr in subsequent experiments. The standard curve drawn with AHG in a PB is indicated in Fig. 3. RLS was proportional to the concentration of AHG within a range below 200 jg/ml. The anti-AHG antisera reacted little to NHG and normal human sera. The standard curve with AHG in normal human serum differed slightly from that in PB (Fig. 4). Immunoglobulin class ofrabbit antisera. The reaction ofAHG with the IgG and IgM fractions of the rabbit antibody against AHG was measured by nephelometry, with the results shown in Fig. 5. The IgG antibody reacted strongly with AHG. The reaction with unfractionated antiserum formed a curve similar to that given by the IgG antibody. The IgM fraction showed little binding activity to AHG. The IgG fraction also reacted with AHG in immunodiffusion. The IgM fraction, however, failed to react with AHG. CIC in healthy subjects andpatients. As shown in Fig. 6, immune complexes ofless than 15 ,ug/ml were seen in sera ofmore than 90% ofclinically healthy subjects, the cases ofhigher titre being found in various diseases. These results generally conform to the analyses reported by many researchers (Lurhuma, Riccomi & Masson, 1977; Cano et al., 1977; Thomas et al., 1978; Brohee et al., 1978; Cambiaso et al., 1978).

Fig. 1. The precipitin pattern in Ouchterlony tests. (1) NHG 1 mg/ml, (2) AHG 1 mg/ml, (3) NHS, (4) anti-AHG, (5) anti-human IgG. 544 K. Arai & A. Shimizu

80 /)- 70 4? I.. 60 > 50 0O 40 -r: 30 20 10 ,1 2 3 4 "20 Time (hr) jug/ml Fig.2 Fig.3 Fig. 2. Time course of the reaction of AHG to anti-AHG at room temperature. Fifty microlitres of AHG (200 pg/ml), 500 p1 of anti-AHG serum (diluted x 30) and 450 p1 of PB containing 20 mM EDTA were mixed.

Fig. 3. Standard curves for CIC quantitations. Reaction ofthe AHG in PB with anti-AHG serum was measured by laser nephelometry. (a) Heat-aggregated human IgG, (b) native human IgG.

a)

10 50 100 2'00 300 153065 125 250 L9 /Ig/ml FFig. 4 Fig. 5 Fig. 4. Comparison of the reaction of AHG and anti-AHG in PB (a) and that in NHS (b).

Fig. 5. The reaction of fractionated antibody and AHG. Five hundred microlitres of IgG fraction (a), IgM fraction (b) and original serum (diluted x 30) (c) were mixed with 500 pI ofAHG solution ofeach concentration. The binding was measured with a nephelometer.

130 120

a) 110

0 100 90 a) I 80 'I E 70 0 or 0 LLJ 60 50 :3 -I E 40 E 30 20 -1 10 -II rI - I---@1i-- NH SLE RA JRA PSS ITP APA LC Hash Gr Fig. 6. Levels of immune complexes in sera from patients with various diseases and from control subjects. NH = normal human, SLE = systemic lupus erythematosus, RA = rheumatoid arthritis, JRA =juvenile rheu- matoid arthritis, PSS = progressive systemic sclerosis, ITP = idiopathic thrombocytopenic purpura, APA = aplastic anaemia, LC = liver cirrhosis, Hash = Hashimoto's disease, Gr = Graves' disease. Immune complex assay 545 DISCUSSION

The antisera obtained by immunization with AHG usually react with both AHG and NHG. Although antisera specific to AHG may be obtained by absorption of antisera with NHG, the immune complexes newly formed in absorbed sera may cause disturbance to the assay. Moreover, the antibody activity of absorbed sera was low. Prior inoculation of massive doses of NHG followed by immunization with AHG could produce antisera that react with AHG but not with NHG. By the procedure reported in this paper, it might be possible to develop the antibody specific to conformationally altered immunoglobulin of other classes, which would be useful for measuring CIC of different classes or subclasses. Assay of CIC by laser nephelometry was not disturbed by a trace of anti-NHG which was presumably contained in the rabbit sera. The multivalent antigens, such as immunoglobulin, interact with the multivalent antibodies to form large lattices which are detectable by laser nephelometry. The lattice formation is well known to be dependent on the ratio of antigen and antibody. In the assay system described in this paper, the titre of anti-AHG was adjusted to detect CIC in the range of 5-200 pg/ml. NHG in test sera (10 mg/ml) was apparently in excess ofthe range of antigen as compared with a trace amount of contaminated anti-NHG. RLS was not proportional to AHG concentration within the range of high antigen concentration. Analyses of two different dilutions of test sera are required in order to avoid any error that might arise from excessive antigen. There was a slight difference between the standard curve for the assay with AHG in PB and the standard curve with AHG in NHS as shown in Fig. 4. The complement components and endogenous rheumatoid factor (RF) were considered to confuse the reaction. The influence of complement components might be avoided by using EDTA. AHG added in NHS reacts with endogenous RF which is contained in small amounts in normal sera and which is detectable by highly sensitive methods. The standard curve with AHG in NHS may be shifted occasionally from the curve with AHG in PB, presumably due to the formation of AHG-RF complexes in the test-tubes. Some test sera may contain free RF and RF bound to CIC. As CIC in test sera are saturated by RF at a constant rate, it is to be expected that free RF in test sera interferes little with the reaction of anti-AHG and CIC. In fact, the sera from patients with high RF titres presented various CIC values. The CIC level determined by the method proposed in this paper was not correlated with the titre of endogenous RF. It is presumed that the specificity of antisera prepared by the present procedure is not identical to that of endogenous RF and that RF does not interfere with the assay system. However, realizing the necessity of further careful investigation into the antigenic determinants recognized from the anti-AHG sera, this study is now in progress in our laboratory. The nephelometric assay with anti-AHG sera was compared with the method of solid-phase Cl q binding. While AHG at each concentration in PB was observed at values almost corresponding to each other, the values for CIC in some of the patients tested by our system did not correspond to the values determined by the Cl q-binding assay. The difference in saturation ofimmune complexes with endogenous RF or complement components among sera may be a possible explanation of the discrepancies in the data produced by these two methods. It is possible that such discrepancies are detected at a particular stage of some diseases. It is necessary to measure CIC simultaneously by several methods using different reagents (Cairns, London & Mallick, 1980). Levinsky & Soothill (1977) have used a low-affinity rabbit IgM anti-human native IgG reagent for detection of CIC. According to these researchers, the IgM antibody is more reactive to polymeric IgG than to monomeric IgG by virtue of the multivalence of low-affinity IgM antibody, having a high avidity for aggregated or antigen-complexed IgG. The rabbit antibody against AHG prepared by our procedure is of IgG class, not containing much IgM. The rabbit IgG antibody against AHG proved in this experiment to react strongly to AHG but not to native human IgG. The antibody prepared by the immunization protocol described in this paper is considered to possess a specificity to conformationally altered IgG in immune complexes. The immune responses to the antigenicity common to aggregated IgG and native IgG were suppressed by the prior intravenous injections ofa large dose of native IgG. The IgG antibody that 546 K. Arai & A. Shimizu specifically reacts to AHG is superior to the low-affinity IgM antibody against native IgG for the purpose of CIC assay by nephelometry. Such an IgM antibody may react with native IgG which is contained in a high level in the test serum and may not help the accurate analysis of CIC. Henney & Ishizaka (1968) have prepared anti-AHG antibody from the guinea-pig by immunizing with AHG and absorbing the guinea-pig serum with native human IgG. This absorbed serum might contain soluble immune complexes, which may interfere with the nephelometric assay of CIC.

The authors wish to express their profound gratitude to Drs T. Hamaoka and S. Shigemoto ofOsaka University Medical School for their valuable advice on the preparation of specific antisera, and also to Professor K. Miyai of their own laboratory for his guidance and encouragement. CIC was assayed by the Cl q-binding method by courtesy of the Special Reference Laboratory, Tokyo.

REFERENCES

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