Flow-cytometric detection of circulating immune complexes

In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.     

An enzyme-linked immunoassay for circulating immune complexes using solid phased goat Clq

An ELISA procedure was developed for measuring circulating immune complexes (IC), using solid phased goat Clq. The use of purified Clq from this species significantly diminished the background uptake of the enzyme-labeled goat antibodies used in the assay, in comparison with Clq isolated from human, guinea pig or rabbit serum. The test specimen results are reported as micrograms equivalent (microgram eq)/ml of heat aggregated human immunoglobulin G (HAIgG), and are based on standard curves developed with this latter reagent for each assay. The 2 World Health Organization (WHO) reference preparations for immune complex determinations (HAIgG and a human tetanus antitoxin-toxoid immune complex) were assayed by this ELISA procedure, and the results obtained were in very good agreement with the WHO established values. All the reagents in the ELISA, including the lyophilized HAIgG standard and the solid phased Clq are stable for more than one year at 4 degrees C. The range of accurate quantitation in test serum is 2-500 micrograms/ml, when using a 1:100 specimen dilution. The total incubation is less than 2 h, with no preliminary preparation of test specimens. The average concentration of IC reactivity in 126 healthy adults was 6 micrograms eq/ml, and the normal upper limit was determined to be 12 micrograms eq/ml.     

HIV antigen detection in circulating immune complexes

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.     

Solid-phase enzyme immunoassay for detection ofChlamydia trachomatis

The prototype of a solid-phase enzyme immunoassay for the detection ofChlamydia trachomatis antigen was tested in 403 men and 135 women attending a venereal disease clinic. Culture on HeLa 229 cells was used as reference method. In men the overall sensitivity and specificity of the enzyme immunoassay was 70 % and 98.5 % respectively. In the 158 men with non-gonococcal urethritis the sensitivity and specificity was 73 % and 96.5 % respectively, in the 31 men with post-gonococcal urethritis 82 % and 100 %, in the 48 men with gonococcal urethritis 67 % and 100 %, in the 125 men with discharge but normal urinary sediment 54 % and 99.1 % and in the 41 men with no signs or symptoms 50 % and 100 %. In women the sensitivity and specificity of the assay was 70 % and 92 % respectively. On account of the low degree of sensitivity in both men and women and the low degree of specificity in women the prototype of the enzyme immunoassay is not yet considered suitable for clinical use. An improved model is being developed.     

Detection of circulating immune complexes with a Raji cell enzyme immunoassay

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.     

The detection of circulating immune complexes in renal transplant patients.

The Raji cell assay and radiolabelled Clq binding method were used to detect circulating immune complexes in the sera of renal transplant patients. Complexes were found in seven of twelve patients using the Raji cell assay; only one serum sample was positive by the Clq method. In five patients the complexes were detected prior to the clinical diagnosis of rejection and in those in whom treatment reversed the rejection the complexes rapidly disappeared. The presence of complexes correlated with a vascular type of rejection characterised by fibrin deposits in the glomeruli in the absence of immunoglobulin or C3 deposits. In two patients, in whom anti red cell antibodies were present, irreversible rejection occurred without the presence of detectable complexes in the sera.     

A rapid solid phase assay for the detection of circulating immune complexes

A simplified process, which we have termed Enzyme Immune Complex Assay (EICATM) for the detection of circulating immune complexes (CICs), is described herein. The method utilizes readily available reagents, small quantities of serum, and can be performed quickly with a minimal amount of equipment. Serum from 38 normal controls, 98 burn patients, 36 frostbite injury patients, and 21 patients with elevated rheumatoid factor (RF) were tested for CICs in an immune function study. Elevated immune complex levels were found in the group of patients with frostbite injury, and in the group with elevated RF. Serum from thermally injured patients had slightly depressed yet normal CIC levels. The detection of elevated CICs by the EICATM method compared favorably with the more cumbersome Raji cell method, with the added advantage of simplicity, speed, and the ability to detect non-IgG immune complexes.     

Chronic liver disease: the detection and characterization of circulating immune complexes.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.     

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab')2 antibodies that caused false positive results when the F(ab')2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab')2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.     

Solid-phase immune electron microscopy-double-antibody technique for rapid detection of papovaviruses.

The solid-phase immune electron microscopy-double-antibody technique, which takes less than 1 h to perform, was applied as a rapid, sensitive, and specific diagnostic tool in the demonstration of papovavirus particles. BK virus propagated in 82C human skin fibroblasts and a monospecific high-titer immune serum to BK virus were used to establish the test procedure. When Formvar-carbon-coated grids were treated with appropriately diluted antibody, a 28-fold increase of virus particles per square micrometer was observed. Viewing of the virus particles was facilitated by the addition of a second "decorator" antibody. BK virus preparations at concentrations of 10(2) to 10(3) PFU/ml could be detected by this technique. There was no cross-reaction with mouse polyomavirus.     

Solid-phase enzyme immunoassay for Chlamydial antibodies.

An enzyme immunoassay (EIA) for chlamydial immunoglobulin G antibodies was developed by using microtiter wells coated with partially purified reticulate bodies of Chlamydia trachomatis serotype L2, grown in McCoy cells, and uninfected McCoy cells as a control. Duplicate testing of a single serum dilution, 1:500, was found to be sufficient. A good correlation between positive reactions was observed in a comparative study of 421 patient sera with the EIA and an inclusion immunofluorescence test. A good correlation between positive reactions was also observed in a comparative study of 140 patient sera with EIA and microimmunofluorescence tests in which chlamydial elementary or reticulate bodies were used as antigens. Sera of 77 healthy control individuals with low titers in inclusion immunofluorescence or complement fixation tests gave negative results in the EIA. Immunoblotting experiments showed that the major antigenic component in the EIA antigen was a protein with an Mr of 39,000.     

Evaluation of a Cordia-IC enzyme-linked immunosorbent assay kit for the detection of circulating immune complexes.

A commercial kit (Cordia-IC) from Cordis Laboratory, Miami, Fla., was compared with the Raji cell radioimmunoassay for its ability to detect circulating immune complexes (CIC) in sera from 30 control subjects and 118 patients with infectious diseases. The 118 patients were categorized into the following groups: (i) 23 patients with bacterial endocarditis, (ii) 41 patients with bacteremia from an infected intravascular catheter or access device, and (iii) 54 patients with Staphylococcus aureus bacteremia related to a deep tissue infection. The Cordia-IC was comparable to the Raji cell radioimmunoassay in intraassay variability (4.0 versus 8.0%) and interassay reproducibility (8.7 versus 20.0%). Neither assay found CIC amounts above 12.5 micrograms equivalents (eq) of aggregated human gamma globulin (AHG) per ml in any of the 30 control individuals. In group 1, Cordia-IC detected 19 of 23 positives (mean, 73.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 23 positives (mean, 54.8 micrograms eq of AHG per ml). In group 2, Cordia-IC detected 19 of 41 positives (mean, 20.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 41 positives (mean, 15.1 micrograms eq of AHG per ml). In group 3, Cordia-IC found 38 of 54 positives (mean, 28.0 micrograms eq of AHG per ml), whereas the Raji cell found 32 of 54 positives (mean, 23.9 micrograms eq of AHG per ml). Statistically, these findings were not significantly different in any of the three patient groups (P> 0.15), and there was an overall good correlation between the results obtained by the two assays (r+0.64, P<0.001). The Cordia-IC provided a suitable assay for the detection of CIC and might find application in routine clinical laboratories.     

Analysis of circulating IgA and detection of immune complexes in primary IgA nephropathy.

The sera of 31 patients with primary IgA nephropathy were investigated for IgA containing immune complexes by Raji cell-binding IgA radioimmunoassay and conglutinin-binding IgA radioimmunoassay. Positive results, without correlation with IgA serum levels, were found in 68% of the patients using the first assay, in 39% of the patients with the second assay. Positive sera were analysed by gel chromatography. Conglutinin-binding IgA eluted in two peaks, a minor one of 400,000-800,000 daltons mol. wt and a major one corresponding to monomeric IgA. No increase of secretory IgA and of polymeric IgA was detectable. IgA immune complexes were likewise found in the sera of patients with systemic lupus (five of 12), rheumatoid arthritis (four of 12), subacute bacterial endocarditis (four of 12) and HB virus hepatitis (four of 16). However, the high prevalence on these sera of IgG and IgM immune complexes detected by polyethylene glycol precipitation, solid phase Clq binding assay contrasted strongly with their absence in IgA nephropathy. In addition, the presence of abnormal amounts of conglutinin reactive IgA correlated with the recurrence of IgA deposits after renal transplantation (20 patients studied). Conglutinin reactive IgA could contribute to the glomerular deposition of IgA and subsequently play a significant role in the pathogenesis of IgA nephropathy.     

Hepatic uptake of circulating IgG immune complexes.

IgG antibodies were found to increase the uptake of circulating dinintrophenylated human serum albumin (DNP-HSA) preparations by the nonparenchymal liver cells in rats. Highly DNP-conjugated HSA was taken up by the Kupffer cells both when given alone and when complexed by IgG. More lightly DNP-conjugated HSA was taken up mainly by the liver endothelial cells. Here, IgG promoted the antigen uptake both by the Kupffer cells and by the endothelial cells. Uptake of IgG immune complexes (IgG-ICs) by the sinusoidal endothelial cells of the liver is a new aspect on the function of these cells. Whether or not this phenomenon is Fc receptor-mediated is discussed. A heat-labile serum factor was found to direct the ICs to the Kupffer cells. This implies that serum complement and hepatic C3 receptors are essential for the physiological clearance of circulating immune complexes.     

Evidence for circulating immune complexes in sarcoidosis

Immune complexes were detected by the platelet aggregation technique in sera of six out of twenty-six patients with sarcoidosis. Five of these patients had acute bilateral hilar lymphoma syndrome, four of them with concomitant erythema nodosum. The size of the immune complexes was 19S or larger.     

Solid phase C1q microassay for the rapid detection of circulating immune complexes

A C1q solid phase microassay was designed for the rapid detection of circulating immune complexes. Its level of sensitivity is comparable to that of the Raji cell and greater than the C1q binding assay; furthermore, it is faster and low in cost. These conditions make it more practical and applicable in the clinical setting.