Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.     

C1q has albumin binding activity but does not interfere in the hemagglutination test for anti-albumin antibodies

Two factors in the sera of patients with liver disease interact with polymerized human serum albumin (HSAP). These are anti-albumin antibodies (AAA) and receptors for polymerized albumin on HBsAg. Recently it was demonstrated by radioimmunoassay that purified human C1q also binds HSAP. Our data confirm the reaction between purified human C1q and HSAP by passive hemagglutination and by indirect immunofluorescence of HSAP-coated erythrocytes (E-HSAP). It is shown that although purified C1q binds HSAP, in the serum, the AAA and not C1q are responsible for the albumin binding activity in HBsAg-negative sera of patients with liver disease and of normal individuals, as detected by passive hemagglutination of E-HSAP, AAA were found to inhibit the binding of serum C1q to polymerized albumin, and hence the E-HSAP hemagglutination test for AAA titration in human sera proved valid.     

An ELISA technique for the measurement of C1q in cerebrospinal fluid

Determination of the C1q content of cerebrospinal fluid (CSF) may be of value in understanding the immunological reactions occurring within the central nervous system (CNS). A double sandwich ELISA method has been developed for the detection of C1q in human serum and CSF. It uses polyclonal antibodies and is sensitive in the nanogram range. The mean concentrations of C1q were determined to be 127 micrograms/ml in serum and 0.4 microgram/ml in CSF. These results suggest that increased levels of C1q in the CSF play a role in some neurodegenerative disorders.     

Porcine C1q and the solid-phase immunoassay of human immune complexes

Experiments were undertaken to determine if porcine C1q could replace human C1q in the solid-phase immunoassay of human immune complexes (ICs). Porcine C1q was obtained by a two-cycle precipitation method involving dialysis against chelating agents in low ionic strength buffer. C1q was adsorbed to polystyrene beads and in vivo- or in vitro-formed ICs binding to the solid-phase C1q were detected with ¹²⁵I-labeled or horseradish peroxidase-conjugated anti-human gamma antibodies. Unfractioned, heat-aggregated human gamma globulin (ΔIgG) could be detected at 20 ng/ml when diluted in buffer only. The detection threshold changed to 40–80 ng ΔIgG/ml when the assay was run with buffer containing normal human serum diluted 1 : 1000 (the serum dilution used for detecting natural ICs). Analysis of systemic lupus erythematosus sera revealed that 60% contained highly significant levels of ICs (binding ?3 S.D. above the mean of controls). Comparison with platelet aggregation test results revealed a highly significant correlation between the two methods (P < 0.0001), even though each assay detected ICs in several serum specimens negative in the other test. These results demonstrate that porcine C1q can functionally replace human C1q in the solid-phase immunoassay of human ICs. Since porcine blood is normally a waste product of the meat-processing industry, it is an obvious source of easily isolated C1q for use in such an assay.     

Current situation and problems in the estimation of circulating immune complexes]

Some methods employing murine monoclonal antibodies have been developed for the estimation of circulating immune complexes (ICs). In the assays using monoclonal antibodies to C1q and C3d, ICs attached by reaction of C1q or C3d with the corresponding antibodies are detected by enzyme-labelled anti-IgG antibody. The murine monoclonal rheumatoid factor (RF) of IgG class is employed in the assay for detection of ICs. ICs reacted with the RF on the solid phase are further detected by the reaction with the second anti-IgG antibody labelled with the enzyme. The anti-C1q antibody in the sera as well as ICs produces positive reactions in the solid phase C1q assay, the assays using monoclonal antibodies are recommended for use in the detection of circulating ICs. In the pretreatment of serum samples, heating at 56 degrees C induces aggregation of IgG to produce a positive reaction by these sensitive assays, and the addition of EDTA-Na2 increases free C1q detached from C1 to induce increased binding to IgG. Reactions of aggregated IgG with RF and C1q in the fluid phase inhibit the following binding of monoclonal RF and anti-C1q antibody on the solid phase. Sera of patients with SLE were examined for CH50, anti-DNA antibody and ICs. The levels of ICs determined by the anti-C1q and C3d antibody assay did not correlate with other parameters. Positivity of ICs was unexpectedly lower in SLE sera. To evaluate the significance of the estimation of ICs, more data must be analyzed by these methods.     

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology

Gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539–684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526–538, aa 590–613 and aa 625–655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.     

Anti-C1q antibodies in hepatitis C virus infection

Autoantibodies against C1q have been described in many immune-complex diseases including hypocomplementaemic urticarial vasculitis and systemic lupus erythematosus (SLE). No study has focused on the role of anti-C1q antibodies in hepatitis C virus (HCV) infection. The aim of this study was (i) to evaluate the prevalence of anti-C1q antibodies in HCV infection; and (ii) to analyse the association of anti-C1q antibodies with clinical and biological features of HCV-mixed cryoglobulinaemia (MC) vasculitis. We searched for anti-C1q antibodies using an enzyme-linked immunosorbent assay (ELISA) test in 111 HCV patients (75 had cryoglobulin and 23 systemic vasculitis), 60 SLE patients and 109 blood donors. Anti-C1q antibodies were detected in 26% of HCV patients compared to 10% of healthy donors (P < 0.01), and 38% in patients with SLE. Although there was a higher prevalence of anti-C1q antibodies among HCV patients with type III cryoglobulin (50%, P < 0.01), the overall prevalence of anti-C1q antibodies was similar in HCV patients being cryoglobulin-positive or cryoglobulin-negative (26%versus 25%, P = 0.98). A significant association was found between anti-C1q antibodies and low C4 fraction of complement (P < 0.05). No association was found between anti-C1q antibodies and HCV genotype, severity of liver disease or with specific clinical signs of HCV-MC vasculitis. This study shows an increased prevalence of anti-C1q antibodies in HCV-infected patients. Anti-C1q antibodies were associated with low C4 levels. No association was found between anti-C1q antibodies and HCV-MC vasculitis, nor between anti-C1q antibodies and cryoglobulinaemia.     

Influence of ionic strength, EDTA concentration, endogenous C1q and polyanions on the 125I-C1q-binding test

Several parameters of the 125I-C1q-binding test were investigated: ionic strength, pH, concentration of EDTA, influence of serum C1q and the possibility of interference by polyanions. Lowering the ionic strength of the borate buffer resulted in increased precipitation of 125I-C1q in normal human serum. This increase was dependent on the presence of serum proteins, probably immunoglobulins. When the concentration of the EDTA was decreased, increased precipitation of 125I-C1q in normal human serum was also observed. This was prevented by adding NaCl to the EDTA solution. However at very low concentrations of EDTA (too low to chelate all calcium ions in the serum), increased precipitation of 125I-C1q in normal human serum was observed even in the presence of added NaCl. Addition of purified C1q to sera from patients with very low C1q levels had varying effects on the results of the C1q-binding test: (a) it decreased the C1q-binding activity of some sera, probably by competition with 125I-C1q for binding sites on the immune complexes; (b) it increased the C1q-binding activity of other sera, probably by enhancing the precipitation of immune complexes as a consequence of the cross-linking effect of C1q; or (c) it had no influence, possibly due to the opposite effects of (a) and (b). The addition of dextran sulphate resulted in a dose-dependent increase in the 125I-C1q-binding activity of normal human serum. This effect was dependent on the interaction of dextran sulphate with either C1q or low-density lipoproteins and was prevented by addition of polybrene to the assay. However, addition of polybrene to sera with a high C1q-binding activity scarcely influenced binding activity.     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

检测可溶性TREM-1的ELISA法的建立及初步应用

目的:建立定量检测可溶性TREM-1的抗体夹心ELISA法。方法:采用抗人TREM-1单克隆抗体(mAb)包被酶标板,以兔抗鼠TREM-1多克隆抗体为夹心抗体、HRP标记羊抗兔IgG为检测抗体、重组小鼠可溶性TREM-1为标准品,建立检测可溶性TREM-1的ELISA法,并对30例正常人和30例急性肺部感染患者血清样本进行了检测。结果:建立的夹心ELISA法检测TREM-1的线性范围为0.78～200μg/L,批内、批间变异系数分别为6.52%和9.46%。30例正常人和30例血清急性肺部感染患者TREM-1的含量分别为(0.69±0.18)μg/L和(1.16±0.42)μg/L,两者比较差异有统计学意义(P<0.001)。结论:成功建立了一种灵敏度高、稳定性好的检测可溶性TREM-1的抗体夹心ELISA法。     

High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.     

Measurement of anti-DNA antibodies by ELISA: a comparative study with Crithidia and a Farr assay

One hundred and twenty six sera from 116 patients with systemic lupus erythematosus (SLE) and from 51 control patients were assayed for the presence of anti-DNA antibodies, using a commercial enzyme linked immunosorbent assay (ELISA). Fifty three sera (42%) from SLE patients were positive and a further 13 sera (10%) fell in the 'equivocal' positive range. Three control sera were positive. In a standard 14C DNA Farr assay, 67 sera (53%) from SLE patients were positive. One control serum was weakly positive. There was a good linear correlation between absorption in the ELISA and the 14C DNA binding result (r = 0.73). Results in the ELISA and Farr assays were concordant in 96 of the 126 SLE sera, and 47 of 51 control sera. Sequential sera from a further 6 patients with fluctuating clinical activity of SLE showed similar patterns of change of anti-DNA antibodies in both assays. The ELISA was more sensitive than the Crithidia luciliae immunofluorescence assay which detected 44 positive sera (35%) in the SLE group. These results suggest that this ELISA assay may be a useful alternative to the Crithidia assay or an effective screen prior to testing in the more technically difficult and time consuming Farr assay for the measurement of anti-DNA antibodies.     

Comparative study of circulating immune complexes quantity detection by three assays--CIF-ELISA, C1q-ELISA and anti-C3 ELISA

The assessment of the soluble immune complexes (IC) in human sera is traditionally performed by the C1q binding assay. In the present study, a novel method for the quantity of immune complexes was reported. The methodology was based on measuring their deposition on solid-phase C3 binding glycoprotein (CIF), using an enzyme-linked immunosorbent assay. We also used ELISA that employed anti-C3 antibodies to determined the quantity of immune complexes. The three assays were evaluated for their performance characteristics on the same specially prepared samples: 55 normal sera, 99 sera from RA, 88 sera from SLE, and 27 sera from PSS. The results were compared by reference to a common standard-heat aggregated IgG that possesses many activities of immune complexes. Three of the tests used displayed almost the same specificity (over 95%), while their relative sensitivity varied depending on the disease sera tested. The sensitivity of the assays used was recorded highest for C1q ELISA-28.97% of positive sera, followed by CIF-ELISA-19.63% and lowest for anti-C3 ELISA-17.29%. A well-expressed correlation was found between CIF-ELISA and anti-C3 ELISA data (r=0.42), and a week correlation was noted when comparing CIF-ELISA and C1q ELISA IC levels detected (r=0.28). When the correlation coefficients were calculated individually for each disease category, they were clearly different, and that reflected indirectly in different sensitivities of the test for various disease categories. We also found that the results from the simultaneous performance of the tests demonstrated low percentage positive results when three or two assays were used. This is most probably due to the different assay abilities to detect IC with different sizes and composition, which shows that a small part of IC in the tested sera can be detected simultaneously by more than one assay. On the basis of the results obtained, we concluded that optimal screening for IC could be achieved by parallel application of several different methods.     

Production of monoclonal antibodies against human erythropoietin and their use in the purification of human urinary erythropoietin

Several murine monoclonal antibodies (MAbs) specific for human erythropoietin (HuEpo) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag.8-653, with the splenocytes of mice immunized with recombinant human Epo (rHuEpo). Based on epitope analysis by a competitive binding assay, these MAbs could be classified into at least three groups: (1) 1E10, (2) 1H7, (3) 2D6, 3D6 and 3D8. In a sandwich enzyme-linked immunosorbent assay (ELISA), using these MAbs as the solid-phase antibodies, MAb-bound HuEpo was detected with rabbit anti-HuEpo sera. Some combinations of two different classes of MAbs, such as 1H7 and 3D8, were found to capture much more HuEpo than each MAb used individually. Urinary HuEpo (U-HuEpo) was highly purified from the urine of patients with severe aplastic anemia with about 50% final recovery using an immunoaffinity column on which a mixture of 1H7 and 3D8 was immobilized. The purified U-HuEpo had a specific activity of 77,340 U/mg in a radioimmunoassay (RIA) and of 76,673 U/mg using an in vivo bioassay.     

McAb夹心ELISA测定人IL-2及其影响因素的探讨

用抗人白细胞间素-2(IL-2)单克隆抗体(McAb)及多克隆抗体建立了敏感的测定IL-2的夹心ELISA。测定范围为1.5u～1000u/ml,与生物学方法有较好的可比性。十二烷基磺酸钠、盐酸胍、尿素及二巯基乙醇等重组IL-2纯化制备中常用的变性剂对本法有不同程度的干扰,而血清则无。用戌二醛预处理包被板可明显减弱这些试剂的干扰作用,并可将测定的敏感性提高2倍。本法也可用以快速确定亲和层析纯化IL-2的条件及抗IL-2McAb的构相表位相关性。     

Autoantibodies against mannose-binding lectin in systemic lupus erythematosus

In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL-complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab')2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.     

Detection of autoantibodies against the globular domain of human C1q in the sera of systemic lupus erythematosus patients

The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients' sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients for the presence of anti-gC1q autoantibodies using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes.     

海洛因依赖者血清中TGF-β1水平的研究

通过检测TGF-β1在海洛因依赖者血清中水平的变化,为进一步认识海洛因对机体免疫功能的影响提供依据。将99例海洛因依赖者按吸食海洛因时间长短均分为三组(2年以内、5~10年、10年以上),33例正常人血清作为对照组,用双抗体夹心酶联免疫吸附试验(ELISA)检测各组血清TGF-β1水平。结果显示,各组海洛因依赖者血清TGF-β1较正常对照组降低(P<0.05);从2年以内组到5~10年组到10年以上组,TGF-β1水平逐渐降低(99.92±38.35 pg/ml、91.72±67.78pg/ml、53.51±27.32 pg/ml)。TGF-β1在海洛因依赖者血清中水平改变明显,吸食海洛因可抑制机体TGF-β1的水平表达。