Critical aspects of immune complex assays employing polyethylene glycol

Treatment of artificial immune complexes (ICs) with 2.5% polyethylene glycol (PEG) — conditions under which C1q-binding activity is routinely measured in the fluid phase — produced marked changes in molecular size as determined by Sepharose 6B chromatography. The effect of PEG on the C1q-binding capacity of ICs, was therefore investigated using a solid phase (SP) system.

PEG enhanced the binding of aggregated human gammaglobulin (AHG) and artificial ICs to SP-C1q and, in reverse experiments, also increased the binding of C1q to SP-AHG. The degree of enhancement varied according to the Ag: Ab ratio employed; the binding of ICs formed in moderate Ab excess was only modestly enhanced but that of complexes formed at slight Ab excess, equivalence and Ag excess was markedly elevated. The profile of PEG-induced enhancement of binding paralleled that of similar ICs in the C1q fluid phase system, suggesting that C1q binding in the latter may be influenced by PEG. However, the C1q-binding activity of in vivo-formed ICs seemed to be relatively unaffected by PEG since enhanced binding was comparable in control and pathological sera.

The results indicate that PEG causes cross-linking and aggregation of ICs (and possibly other serum proteins) which may alter their biological activity and hence influence the results of IC assays that employ this agent.     

Reactivity of low molecular weight material in cellular immune complex assays.

A major difference in molecular size of material reactive in the C1q binding assay and two cellular assays (Raji and L1210) for immune complexes, is reported. Elevated C1q binding of pathological sera was associated with material in the range 7-19S, as determined by Sepharose 6B chromatography of sera from patients with chronic inflammatory and neoplastic lung diseases. By contrast, reactivity of identical sera in the Raji and L1210 assays was linked predominantly with material of molecular size 7S. Dissociation of immune complexes on storage and/or in consequence of the chromatographic procedure was effectively discounted. Furthermore, differential binding of 7S IgG fractions tested at a standard concentration indicated that reactivity in either test was not attributable to non-specific binding of IgG. In a previous study, saturation of FcR (on L1210 and Raji) and C3R (on Raji only) by heat-aggregated IgG failed to distinguish whether binding directly involved these receptors or other cell surface components. In the present investigation, no firm correlations emerged between reactivity in the two tests and possible candidate antibodies reactive with cell surface components such as anti-lymphocyte and anti-nuclear antibodies. It is therefore suggested that low molecular weight binding may be attributable to more than one factor including 7S IgG (immune complex dissociated, or otherwise), autoantibodies, IgG-C3 complexes and possibly very small immune complexes (Ag1, Ab1). The assumption that Raji and L1210 and possibly other cellular assays detect only high molecular weight immune complexes is questionable and the need for further characterization of other reactive material is emphasized.     

Evaluation of two photometers used in enzyme-linked immunosorbent assays.

The performance of two commercially available high-speed photometers, designed for through-the-plate reading, was evaluated. Linearity of instrumental reading and reproducibility of same-day and 2-day measurements were assessed by least-squares analysis and analysis of variance, respectively. For both instruments, the photometric error was on the order of thousandths of an absorbance unit and was much smaller than the error of the currently available enzyme-linked immunosorbent assays.     

Evaluation of rubella immune status by three commercial enzyme-linked immunosorbent assays.

Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (Enzygnost-Rubella, RUBELISA, and ORTHO Rubella) were evaluated for the determination of immune status by testing 1,090 serum specimens, 410 of which were from nonimmune patients. In comparison with the standard reference technique, the hemagglutination inhibition (HAI) test, the sensitivities of ORTHO Rubella (100%) and Enzygnost-Rubella (99.26%) were excellent, whereas the sensitivity of RUBELISA (95.60%) was marginally lower because of the inability of this assay to detect antibody in 22% of the serum specimens with HAI titers of 10 and 11% of sera with HAI titers of 20. The specificity of all three systems was greater than 97%. There was a linear correlation between mean ELISA values and increasing HAI titers (r greater than or equal to 0.94). Both ORTHO Rubella and Enzygnost-Rubella were shown to be suitable replacements for the HAI test, provided that an equivocal zone is incorporated in the ORTHO system and only unheated sera are used in the Enzygnost system.     

Pathogenesis of IgA nephropathy: In vitro activation of human mesangial cells by IgA immune complex leads to cytokine secretion

IgA immune complex (IC) plays a crucial role in the pathogenesis of IgA nephropathy (IgAN). As IgA-IC is not itself cytotoxic, other mediators may be involved in the pathogenesis. In order to elucidate the mechanisms by which IgA-IC mediates renal injury in IgAN, the ability of IgA-IC to ‘activate’ cultured human mesangial cells (HMC) was studied. HMC were incubated with nephritogenic IgA-IC, containing a MOPC-315 plasmacytoma-derived IgA anti-dinitrophenyl (DNP) and DNP-conjugated bovine serum albumin. The cells showed morphological changes, an accelerated rate of proliferation, and increased production of interleukin-1 (IL-1), interleukin-6 (IL-6), platelet activating factor (PAF) and generation of superoxide anion. The enhancement of IL-1 and IL-6 mRNA expression in HMC incubated with IgA-IC was identified by dot blot analysis. Northern blot hybridization also demonstrated an augmented IL-6 mRNA expression in HMC treated with IgA-IC. These results suggest that nephritogenic IgA-IC may amplify the proliferation of HMC and the production of immune/chemical mediators and superoxide anion thereby resulting in the renal lesions of IgAN.     

Characteristics of immune complexes detectable by two independent assays in gynaecological malignancies.

Immune complexes from patients with ovarian and endometrial cancer have been examined using the C1q solid phase (C1qSP) and polyethylene glycol (PEG) precipitation assays. The amount of IgG in the PEG precipitates was inversely correlated with the amount of IgM whilst the IgM values correlated positively with the C1qSP assay values. Separation studies revealed that most of the C1qSP activity was not precipitated from cancer sera by 2% PEG. The immune complexes were fractionated on Sephacryl S-300. Both the PEG precipitation and C1qSP assays detected high molecular weight immune complex like activity (greater than 19S). The C1qSP assay also detected a second peak of activity at approximately 7-8S. Most of the PEG detectable immune complexes from sera dissociated during column chromatography, whereas the C1qSP detectable complexes were relatively stable. Furthermore the IgG from fractionated PEG precipitates emerged as a monomer (7S) component. The PEG assay appeared to be detecting a high molecular weight complex containing mostly IgG rather than IgM with a low affinity for C1q, which was easily dissociated. The C1qSP assay detected a more stable high molecular weight complex containing a relatively high proportion of IgM and a low molecular weight complex, both with a high affinity for C1q.     

Immunoelectron microscopic studies of immune complex deposits and basement membrane components in IgA nephropathy

The relationship between the immune complex deposits of mesangial IgA nephropathy and the basement membrane components, type IV collagen and fibronectin, has been investigated by an indirect immunogold technique in four cases of mesangial IgA disease. Using paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidney, IgA, IgM and C3 were localized in the mesangial electron-dense deposits with 10 and 20 nm gold-labelled secondary antibodies. In the same glomeruli, type IV collagen and fibronectin were rarely present within the electron-dense deposits, although both were distributed throughout the remainder of the mesangial matrix with the exception of the subepithelial regions. These two components were also present within the glomerular basement membrane and localized mainly on the endothelial aspect. A similar distribution of the basement membrane components was seen in a control kidney processed in the same way. This technique gives reproducible results and has demonstrated for the first time the relationship between the mesangial immune complex deposits of mesangial IgA nephropathy and the basement membrane components of the matrix in which they are found.     

Preparation of a defined immune complex for use in C1q binding assays

Soluble immune complexes of human β₂ microglobulin with its IgG antibodies from Macacca irus antisera have been tested for suitability as a reference preparation for immune complex assays in human disease. The defined complex described is stable on storage, behaves reproducibly in a C1q solid phase binding assay and fulfills the criteria required for an international reference standard.     

IgA: an immune glycoprotein

IgA is a glycoprotein containing multiple N-linked carbohydrates as well as O-linked glycans in the case of IgA1. Because of the critical role it plays in providing protection at mucosal surfaces, IgA is an ideal candidate for use as a therapeutic or prophylactic agent. The presence or absence of carbohydrates, as well as their structure, has been found to influence effector functions and binding to specific IgA receptors. In addition, changes in IgA glycosylation are associated with immune pathology. A thorough understanding of the contributions of the glycans to IgA immune protection will aid in the design of clinically suitable antibodies.     

Contrasting roles for tumor necrosis factor in the pathogeneses of IgA and IgG immune complex lung injury.

Recent studies suggest that development of acute gamma G immunoglobulin (IgG) immune complex lung injury is partially dependent on a tumor necrosis factor (TNF)-dependent mechanisms of neutrophil (PMN) recruitment. The authors have sought to further define the role of intrapulmonary TNF in IgG alveolitis and to examine its role in IgA immune complex alveolitis, a neutrophil-independent model of acute lung injury. IgG immune complex lung injury resulted in a marked rise in intrapulmonary TNF activity accompanied by progressive pulmonary PMN accumulation. Intratracheal instillation of neutralizing concentrations of anti-TNF markedly reduced PMN influx measured at 4 hours but had no effect on PMN recruitment quantitated at 2 hours. IgA immune complex deposition resulted in acute lung injury accompanied by increased numbers of intrapulmonary mononuclear phagocytes but few neutrophils. Lung lavage fluids obtained from IgA immune complex-injured rats contained both neutrophil and monocyte chemotactic activities, albeit at twofold to fourfold lower concentrations than observed in IgG-mediated alveolitis. In contrast to IgG complex-mediated alveolitis, lung lavage fluids from IgA-injured rats contained no TNF activity. Intratracheal administration of anti-TNF antibodies had no effect on the development of IgA lung injury as assessed by morphology and measurements of vascular permeability. In vitro exposure of isolated alveolar macrophages to performed IgG immune complexes resulted in dose-dependent TNF secretion, while exposure to IgA complexes resulted in very low levels of TNF secretion. These data suggest that TNF-mediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest chiefly in the late phase (approximately 4 hours) of developing alveolitis. The virtual absence of intrapulmonary TNF activity in evolving IgA immune complex alveolitis may in part account for the limited PMN recruitment observed in this model.     

左旋聚乳酸可吸收骨钉两种灭菌法效果评价

目的评价左旋聚乳酸可吸收骨钉经环氧乙烷灭菌和^60Co-γ辐照灭菌的效果。方法将左旋聚乳酸可吸收骨钉放人环氧乙烷灭菌器中灭菌,灭菌后将材料置于空气中静置5d,以清除残留的环氧乙烷;另将左旋聚乳酸可吸收骨钉采用^60Co-γ辐照灭菌装置灭菌,吸收剂量15kGy。然后对2种方法灭菌的骨钉分别进行性能表征。结果与左旋聚乳酸可吸收骨钉初始弯曲强度相比,经2种方法灭菌的骨钉弯曲强度均无明显下降（P〉0．05）;与左旋聚乳酸可吸收骨钉初始分子量相比,经^60Co-γ辐照灭菌的骨钉分子量明显减小,下降约11％,表明材料发生降解;经环氧乙烷灭菌的骨钉分子量无明显变化,下降约3％,表明材料未发生明显降解。结论左旋聚乳酸可吸收骨钉以环氧乙烷灭菌为佳。     

Circulating IgA immune complexes in aids

Circulating immune complexes were isolated from the serum of patients with AIDS, as well as patients with other acute and chronic viral diseases. Analysis of these immune complexes by methods of flow cytometry and by radioimmune (Raji cell) assay revealed a prevalence of IgA complexes in the serum of AIDS patients and a prevalence of IgG complexes in the serum of patients with other viral diseases. Raji cells bind immune complexes via Fc and complement (C,) receptors and may detect IgA immune complexes more efficiently than a Clq assay since IgA has no affinity for Clq.     

Antibodies to polyclonal IgA,IgA1, and IgA2 and isotype-specific immune complexes in IgA nephropathy

The concentrations of serum IgG and IgM antibodies to polyclonal IgA (IgA_p), IgA₁, and IgA₂ were determined by enzyme immunoassay in 31 patients with IgA nephropathy and 30 healthy controls. Patients with IgA nephropathy had significantly raised concentrations of serum IgA compared to controls (Mann-WhitneyU test,P=0.001) and increased concentrations of conglutinin-binding IgA immune complexes (P=0.024). No differences in the median concentrations of IgG and IgM anti-IgA antibodies were found between the patients and the controls. In serum samples from healthy controls there was a significant positive correlation between IgM anti-IgA_p and IgA immune complex concentrations (P=0.05), which contrasted with the finding of an inverse correlation between IgM anti-IgA_p and IgA immune complex concentrations in patients with IgA nephropathy (P<0.05). In addition, the concentrations of conglutinin binding IgM immune complexes in serum were found to correlate with the concentration of IgM anti-IgA_p (0.010<P<0.025), IgM anti-IgA₁, and IgM anti-IgA₂ (P«0.005 for both) in patients with IgA nephropathy but not in controls. IgM anti-IgA antibodies may be important in augmenting the clearance of IgA immune complexes from the serum of patients with IgA nephropathy.     

Circulating immune complexes in IgA deficiency

Circulating immune complexes (IC) were demonstrated in patients with serum IgA deficiency. Sixteen of thirty-one IgA deficient patients had serum IC detected by solid phase C1q radioimmunoassay for IgG class complexes. The presence of cryoglobulins (thirteen out of thirty-one patients) and increased polyethylene glycol precipitation (ten out of thirty patients) provided additional evidence for the presence of IC. Fourteen patients were asymptomatic but seven had clinical evidence of disease which could have been IC mediated: two with glomerulonephritis, three with polyarthritis, one with vasculitis and one with thyroiditis. Serum IC remained detectable in multiple samples over several months but this correlated poorly with the presence or absence of disease. Serum antibody to IgA was detected in fifteen out of thirty-one patients. There was no direct relationship between the presence of IC and the level of serum anti-IgA antibody; however, this antibody was shown to be present in the IC isolate in eight patients. It is proposed that a considerable portion of the IC load in IgA deficiency results from defective antigen exclusion at the level of the mucosa.     

Complement-mediated solubilization of rat IgA immune precipitates

The complement-mediated solubilization (CMS) of immunoprecipitates (IP) consisting of DNP-rat serum albumin (RSA) and rat monoclonal anti-DNP antibodies of the IgA [both polymeric (p-) and monomeric (m-)] or IgG2b (sub)class was studied. In contrast to IgG2b IP, solubilization of IgA IP was only observed in an autologous system, with rat serum as the source of complement. IP prepared using m-IgA were solubilized faster than those prepared using p-IgA. Analysis of both affinity and avidity of the antibodies, indicated that this difference may be due to the lower avidity of the m-IgA antibodies as compared to p-IgA. Analysis of the solubilized IP revealed deposition of C3 and C4 on IgG2b, and only C3 on IgA IP. These results point toward a role of the alternative pathway in the solubilization of IgA IP. Size analysis of the solubilized IgA IP employing sucrose density gradient ultracentrifugation, indicated that these were heterogeneous, with a size generally larger than 19 S.     

Evaluation of three commercial enzyme-linked immunosorbent assays and two latex agglutination assays for diagnosis of primary Epstein-Barr virus infection.

Three commercially available enzyme-linked immunosorbent assays (ELISAs) from Gull, Biotest, and Behring (Enzygnost) and two latex agglutination tests for heterophile antibodies (Monolatex [Biotest] and Mono-Lex [Trinity Laboratories]) were evaluated for the diagnosis of primary Epstein-Barr virus (EBV) infection and EBV seropositivity. Two hundred fourteen consecutive samples from 197 patients with symptoms of primary EBV infection were analyzed by the five assays at a clinical microbiology laboratory. The samples were also analyzed independently by immunofluorescence methods at a reference laboratory. According to the reference methods, 37 patients (40 serum samples) had primary EBV infections, 120 patients (127 serum samples) had had past EBV infections, 33 patients (36 serum samples) were seronegative, and 7 patients (11 serum samples) exhibited atypical reactions. The respective sensitivities and specificities for the diagnosis of primary EBV infection were 95 and 100% for the Gull assays, 100 and 94% for the Biotest assays, and 100 and 89%, for the Enzygnost assays. The Monolatex and Mono-Lex methods showed similar sensitivities and specificities (78 to 85% and 100 to 99%, respectively) for the diagnosis of primary EBV infection. This study demonstrates the usefulness of commercially available assays for the rapid diagnosis of primary EBV infection, but also the importance of large-scale testing of routine samples before choosing an assay.     

Detection of bovine serum albumin in the circulating IgA immune complexes of patients with IgA nephropathy

Alimentary antigenic challenge has been postulated to have a role in the genesis of IgA circulating immune complexes (CIC), resulting in mesangial IgA disease. In this study, we examined the relationship between bovine serum albumin (BSA) and IgA CIC in patients with IgA nephropathy. Of the 47 patients studied, elevated IgA CIC levels were found in 32% by the F(ab')2 anti-C3 and Raji cell enzyme immunoassays (EIA). Elevated IgA anti-BSA antibody levels were found in 9 patients, and there was a positive correlation between these levels and IgA CIC as measured in the Raji cell EIA (R = 0.60, P less than 0.001). In 4 patients with elevation of both IgA CIC and IgA anti-BSA antibody levels, solubilization experiments were done to demonstrate the presence of BSA antigen in the IgA CIC. Using the Raji cell EIA, the IgA CIC levels decreased significantly after preincubating the sera with serial concentrations of excess BSA. No corresponding effect was seen with human serum albumin used as control. Hence, BSA may be the antigenic stimulus in the formation of IgA CIC in selected patients with IgA nephropathy. The pathogenic capacity of these IgA-BSA CIC remains to be determined.     

C1q binding and Raji immune complex assays: a comparison using defined immunoglobulin aggregates

Aggregated IgG is frequently employed as a standard in systems for the measurement of immune complexes in man and animals. In this paper aggregates prepared by heat or alkali denaturation of human IgG were fractionated by column chromatography through LKB AcA 22 Ultrogel. Heat aggregation yields preparations containing considerably more monomer than alkali treatment (47% and 6.3% respectively). The bulk of aggregated material prepared by both methods was of size 19 S or greater. Smaller aggregates were present in assayable quantities only in the alkali aggregated material. The sized fractions of aggregates IgG were tested in the presence of a human complement source for their efficiency in the C1q binding and Raji radioimmunoassay for immune complexes. Both techniques efficiently measured large aggregates (greater than or equal to 19 S) but the C1q binding assay measured smaller material with greater efficiency than did the Raji cell assay. Neither technique detected monomeric IgG. The date presented is relevant to the binding characteristics of the 2 assay systems studied and suggests that when used together they are capable of measuring immune complexes present over a wide range of sizes.     

Evaluation of two new assays for determination of serum fibrinogen/fibrin degradation products

Two new commercial assays for the detection of degradation products of fibrinogen/fibrin (FDP) were evaluated against two standard procedures. The first, a new hemagglutination inhibition (HAI) assay using glutaraldehyde-treated cells, was compared with the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Analysis of 43 samples from patients with a variety of bleeding disorders and thrombotic conditions showed a high degree of correlation between methods (r = 0.934). The second new assay, a rapid slide test using antibody-coated latex particles, was compared with results obtained by electroimmunoassay. There were no significant differences in the results as assessed by two statistical parameters. It was concluded that both new tests are useful for routine use in clinical laboratories.