A Simple Quantitative Assay of Circulating Immune Complexes by Laser Nephelometry, Using a Rabbit Igg Antibody Against Human Aggregated Igg
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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.