A Solid-Phase Radioimmunoassay for Anti-Histone Antibodies in Human Sera: Comparison with an Immunofluorescence Assay

A solid-phase assay for anti-histone antibodies is described, in which antibodies bound to calf thymus total histones, specific histone classes, or chromatin were detected by radioactive anti-human IgG, IgA or IgM. The reactivity of human sera from various rheumatic diseases was analysed by this assay and compared with results obtained from an indirect immunofluorescence assay (FANA) previously described. Sera that were positive in the FANA assay were usually positive in the solid-phase assay when total histones were the solid-phase antigen. However, many sera had IgM antibodies to total histones in the solid-phase assay but gave no detectable reaction in the FANA assay. Results from the solid-phase assay, using individual histones and chromatin, showed that the anti-histone antibodies in these sera were predominantly reactive with histones H3-H4 and did not bind well to histones complexed to DNA in the form of chromatin.     

The Attachment of Serum-and Plasma-Derived C3 to Solid-Phase Immune Aggregates and its Relation to Complement-Mediated Solubilization of Immune Complexes

The interaction between immune aggregates and complement (C) was investigated. Solid-phase immune aggregates were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA antibody. The immune aggregates were reacted with human serum or citrated plasma at 37 degrees C. The binding of C3 components was investigated with biotinylated F(ab')2 antibodies to C3c and C3d and avidin-coupled alkaline phosphatase. The form of the incorporated C3, whether C3b-iC3b or C3dg, can be deduced from the response with these two antibodies. The maximal binding of C3b-iC3b to the immune aggregates was observed within 5 min of incubation with serum or citrated plasma. The conversion to C3dg was evident by a decrease in bound anti-C3c concomitant with increasing anti-C3d reactivity within about 10 min of incubation. When the classical C pathway activation was inhibited, the binding of C3b-iC3b was delayed by 20-30 min, whereas stopping of the alternative pathway did not influence the initial kinetics of the reaction. The addition of human red blood cells had no measurable influence on the degradation of bound C3b-iC3b. 125I-labelled anti-BSA antibody bound to the solid-phase BSA was not released during the C3 incorporation. The incorporation of C3b into the immune aggregates was mediated equally well by serum and by citrated plasma. The incorporation of C3b-iC3b into immune complexes (IC) is thought to be responsible for the C-mediated solubilization (CMS) of IC. Citrated plasma, however, exerted no CMS capacity when measured by a radiometric assay. The CMS capacity of serum was inhibited by citrate, but could then be restored by adding Ca2+ and Mg2+, whereas no CMS could be demonstrated with citrated plasma to which divalent metal ions were added.     

Solid-Phase Radioimmunoassay for Detecting Bovine (Neonatal Calf Diarrhea) Rotavirus Antibody

An indirect solid-phase microradioimmunoassay is described for detecting antibodies against rotaviruses. The test involved ethanol fixation of microcultures of bovine rotavirus-infected BSC-1 cells and reaction with bovine antirotavirus serum, followed by ¹²⁵I-labeled rabbit anti-bovine immunoglobulin G. The technique was shown to be virus specific and highly sensitive. The fixed microcultures could be stored at 4°C for at least 2 months without affecting the sensitivity of the test. The application of this system for the detection of rotavirus antibodies in humans is briefly discussed.     

Development of a Solid-Phase Radioimmunoassay for the Determination of Arginine-Vasopressin in Urine

A new solid-phase radioimmunoassay (RIA) has been developed for measuring arginine-vasopressin (AVP) in urine. AVP is first extracted from urine by adsorption on Vycor glass powder and eluted with acetone-water (60:40). The mean recovery is 75.3 ± 2.2% (n =18). The organic extract is evaporated to dryness and reconstituted in the assay buffer. Aliquots of this extract are then incubated with ¹²⁵I-AVP in polystyrene LKB tubes previously coated with the antiserum (1:50000) for 48 hours. The free radioactive fraction is removed by aspiration and the tubes are counted. Values correlate well with those obtained by liquid-phase RIA using dextran-charcoal. Urinary AVP concentrations in normal Sprague-Dawley rats and rats with varying degrees of hydration have been measured.     

Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A Antigen in Stool and Antibody to Hepatitis A Antigen in Sera: Comparison with Solid-Phase Radioimmunoassay, Immune Electron Microscopy, and Immune Adherence Hemagglutination Assay

Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.     

Binding of Properdin to Solid-Phase Immune Complexes: Critical Role of the Classical Activation Pathway of Complement

The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme-linked immunosorbent assay using solid-phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake was hardly detectable. Alternative pathway-mediated deposition of C3 with slow kinetics was demonstrated in C2-deficient serum and in NHS depleted of C1q, factor D and properdin (C1qDP-depleted serum) after reconstitution with factor D and properdin. Efficient uptake of properdin required a functional classical pathway, in the presence of which C3 and properdin were rapidly deposited onto the IC. Judging from findings in C3-deficient serum, factor I-deficient serum, and C1qDPB-depleted serum, the uptake of properdin was strictly C3-dependent, and did not require the presence of factors B and D. Thus, C3b fixed to IC was the principal ligand for properdin in the assay. The findings could have biological implications relating to complement-mediated modification of immune complexes in disease.     

The Affinity of Soluble Immune Complexes for Concanavalin A

Con A-Sepharose affinity chromatography may be used in the analysis and classification of immune complexes. Experiments with model immune complexes suggest that the degree of affinity of an immune complex for Con A-Sepharose is determined by the antigen rather than the IgG antibody of the complex. It is possible that partial characterization of unknown antigens linked to IgG in immune complexes may be achieved in many diseases. Preliminary explorations with selected human sera indicate that the IgG containing immune complexes in Burkitt's lymphoma and nasopharyngeal carcinoma have affinity for Con A-Sepharose. By contrast IgG containing immune complexes in chronic hepatitis B seem to lack affinity for Con A-Sepharose.     

Solid-Phase Microtiter Radioimmunoassay Blocking Test for Detection of Antibodies to Escherichia coli Heat-Labile Enterotoxin

The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.     

On the Interaction of the First Complement Component C1 and its Subunit Clq with Solid-Phase IgM Immune Complexes

The interaction of C1 and C1q with solid-phase anti-dextran MOPC-104E IgM was studied. An enzyme-linked immunosorbent assay (ELISA) was used to detect bound C1q. The results revealed that immobilized IgM is converted to the functionally active 'staple' conformation by the specific polyvalent ligand dextran (B 1355/S). C1q is fixed to IgM dependent on the antigen concentration, and its binding might be explained by assuming a functional binding constant (K) of approximately 10(9) M-1. Molecules bound with a K in the range of 10(7) M-1 cannot be detected by this ELISA procedure. The fixation of C1q saturated with an excess of the C1r2S2-tetramer differs from that of free C1q. C1q incorporated in the C1 complex rapidly dissociates independently of the antigen concentration. Since the complement binding sites are located at definite positions on the IgM molecule because of its pentameric structure, it is suggested that the distinguishable association properties of C1 and C1q are brought about from the altered flexibility of the C1q molecule complexed with C1r2S2.     

A Simple Solid-Phase Radioimmunoassay for Morphine and Related Opiates Using A Mixture of Labelled Morphine and Antiserum Pre-Precipitated by Polyethylene Glycol

A novel, solid-phase radioimmunoassay for morphine and reiated opiates is described in which radiolabelled morphine, anti-serum and polyethylene glycol are premixed before being added to the samples or standards. The mixture can be stored at 2 ± C and used for several months until decay of the ¹²⁵I label leads to excessively long counting times. The method was developed using the Roche Abuscreen radioimmunoassay for morphine and has the particular advantages of simplicity (only 2 pipetting steps required instead of 5) and economy (800 assay tubes can be prepared from a 100-tube Abuscreen plus a 50% saving in operator time). The procedure can be applied to other assays providing the polyethylene glycol-precipitated antiserum is stable and that the antibody affinity for the labelled compound is not so high as to prevent displacement of the labelled compound from the antibody binding sites by unlabelled drug.     

Immune Complexes in Human Melanoma: A Consequence of Deranged Immune Regulation

Circulating immune complexes were detected in 62 individuals with malignant melanoma by precipitation with isolated human C1q and polyclonal rheumatoid factors. In 56 patients the C1q deviation assay showed low to moderate levels of complexes, with increased amounts with advancing stage of disease. Both heavy (>19S) and intermediate (7S to 19S) varieties were present, and complexes containing tumor antigen-antibody or antibody- anti-antibody were identified. Complexes were found in the kidneys of one patient with malignancy and the nephrotic syndrome and in two further patients with melanoma in whom there were DO clinical manifestations of nephrosis. Serial determinations in 51 patients showed slow cyclic variations in the levels of complexes and fluctuations in response to therapy. The coexistence of anti-antibodies, immune complex disease, and anergy in melanoma patients may indicate a deranged immune regulation consequent to chronic antigenic stimulation by the tumor.     

A Survey for Circulating Immune Complexes in Patients with Acute Myocardial Infarction

A new assay for the detection of circulating C1q-binding immune complexes (IC) is described. The assay makes use of solid-phase C1q and iodinated soluble protein A, extracted from the cell wall of Staphylococcus aureus. In a model system the assay could detect heat-aggregated IgG down to a concentration of about 50 ng/ml. This method and three other assays, previously described, were used to survey the appearance of IC activity in sera from hospitalized patients with acute myocardial infarction. Depending on the assay system used, from 56% to 66% of the patients investigated were found to develop circulating IC. The earliest appearance of circulating IC was noted 5 days after infarction. The highest incidence of positive reactions and the strongest reactions occurred 2 to 3 weeks after hospitalization; thereafter the IC positiveness tapered off, and all patients were negative 6 weeks after infarction.     

Detection of IgG Aggregates or Immune Complexes Using Solid-Phase C1q and Protein A-Rich Staphylococcus aureus as an Indicator System

A radioimmunoassay for detection of Clq-binding IgG aggregates and antigen-IgG antibody complexes is described The assay makes use of solid-phase Clq and ³²p-labelled protein A-rich Stphylococcusaureus as an indicator system. Both 19S and heavier IgG aggregates that fixed Clq were detected The sensitivity of the assay permitted detection of heavy (19–25S) IgG aggregates at a concentration of 8 μg/ml or less. The results indicated that detection of IgG in this assay is dependent on the degree of IgG polymerization and the molar ratio between the solid-phase Clq and the IgG polymers. Albumin-anti-albumin complexes, preformed at equilibrium with antibody to antigen molar ratios of 2:1 to 3:1 and at antigen concentrations of 25 to 40 μ g/ml. were also detectable using the described radioimmunoassay     

A Solid-Phase Radioimmijnoassay for Bacterial Lipopolysaccharide

A radioimmunoassay for E. coli 055:B5 lipopolysaccharide (LPS) is described. The LPS was derivatised by two new methods and subsequently radiolabeled with 125 I to a specific activity of 2-4 mCi/mg without apparent loss in its biophysical, immunological or biological activities. Using antibody-coated polystyrene tubes, a solid-phase radioimmunoassay was developed with a sensitivity of 10-500 ng/ml of LPS.     

A Solid-Phase Radioimmijnoassay for Bacterial Lipopolysaccharide

A radioimmunoassay for E. coli 055:B5 lipopolysaccharide (LPS) is described. The LPS was derivatised by two new methods and subsequently radiolabeled with 125 I to a specific activity of 2–4 mCi/mg without apparent loss in its biophysical, immunological or biological activities. Using antibody-coated polystyrene tubes, a solid-phase radioimmunoassay was developed with a sensitivity of 10–500 ng/ml of LPS.     

Neutrophil Chemotaxis Induced by Immune Complexes

In the current work we have analyzed the ability of different soluble immune complexes (IC) prepared with IgG antibodies to induce neutrophil chemotactic responses in vitro. While, in all cases, IC were able to induce neutrophil migration in a concentration-dependent fashion, IgG antibodies alone were completely unable to induce locomotor responses. Checkerboard analysis indicated the chemotactic nature of motility. On the other hand, chemotaxis induced by IC was markedly inhibited by IV.3, a monoclonal antibody (mAb) to FcγRII, slightly reduced by 3G8 F(ab′)2, a mAb to FcγRIII, and nearly abrogated by both mAbs. The impact of IC on neutrophil migration induced by FMLP was also studied. We found that when a suboptimal concentration of FMLP was employed, the simultaneous addition of IC increased the migration acting in additive form. The significance of these results is discussed.     

Circulating Immune Complexes in Systemic Scleroderma

Circulating immune complexes were detected by the immunoelectrophoretic method in 18 of 29 (62 per cent) of patients with systemic scleroderma. The presence of immune complexes did not correlate with that of antinuclear antibodies to dsDNA, DNP, RNP, and Sm. The mean levels of immunoglobulins G, A, and M as well as of C3 were significantly higher in patients with systemic scleroderma than in blood donors.     

Circulating Immune Complexes in Systemic Scleroderma

Circulating immune complexes were detected by the immunoelectrophoretic method in 18 of 29 (62 per cent) of patients with systemic scleroderma. The presence of immune complexes did not correlate with that of antinuclear antibodies to dsDNA, DNP, RNP, and Sm. The mean levels of immunoglobulins G, A, and M as well as of C3 were significantly higher in patients with systemic scleroderma than in blood donors.