Raji Cell Radioimmunoassay Research Articles

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.

Lack of Circulating Immune Complexes in Inflammatory Bowel Disease

Multi-organ involvement and especially extraintestinal manifestations have suggested an immune complex-mediated pathogenesis of ulcerative colitis and Crohn's disease. Using various techniques controversial data have been reported on the incidence and levels of circulating immune complexes and their correlation to clinical presentation. Sera of 131 patients with inflammatory bowel disease (78 Crohn's disease, 53 ulcerative colitis) representing a wide spectrum of disease activity, treatment and presence or absence of extraintestinal manifestations were tested for circulating immune complexes using Raji cell indirect immunofluorescence assay, Raji cell radioimmunoassay, C1q solid phase assay and polyethylene glycol precipitation coupled with measurements of optical density and subsequent immunoelectrophoresis or radial immunodiffusion. Circulating immune complexes in low concentrations were observed in a small number of patients with inflammatory bowel disease, the frequency and concentrations being slightly higher in patients with Crohn's disease than in those with ulcerative colitis. No association of concentrations of circulating immune complexes with disease activity or presence of extraintestinal manifestations could be demonstrated. These data do not support the claim for a major role of circulating immune complexes in the pathogenesis of inflammatory bowel disease.

Immune complex-like material in the serum and plasma exchange in patients with metastatic cancer.

Clinical significance of immune complex-like material in the serum was investigated in tumour patients undergoing plasma exchange with albumin-saline solution and subsequent chemotherapy. Immune complexes were detected by the Clq binding assay or the Raji cell radioimmunoassay in nine out of forty-five patients before this therapy. Levels of immune complexes were decreased to 10-30% of the initial value by plasma exchange depending on exchanged plasma volume. In contrast to other serum proteins like alpha 1-antitrypsin and alpha 2-macroglobulin, which showed protein specific increase during follow up after plasma exchange in all patients, recovery rates of immune complexes and IgG were highly individual but parallel in each patient. Clinical response to this protocol did not correlate with immune complex status, suggesting that removal of the measured immune complex like material had little clinical significance or was not longlasting enough to provide therapeutic benefit.

Circulating Immune Complexes in Prosthetic Valve Endocarditis

Using the Raji cell radioimmunoassay, we have determined circulating immune complex (CIC) levels in 36 patients with prosthetic valves during 38 episodes of fever. Fever resulted from prosthetic valve endocarditis (PVE) in 27 instances and from other causes in 11 instances. Peak initial CIC levels higher than 100 micrograms/mL occurred more frequently in the group with PVE, while peak initial CIC values less than 30 micrograms/mL were more frequent in the control group. Circulating immune complex levels fell substantially with completion of antibiotic therapy in 28 (78%) of the patients with PVE. Late CIC elevations were associated with drug-related rashes and replacement of persistently infected prostheses. Our data suggest that the predictive value of measurement of CIC levels in patients with fever and prosthetic valves is in excluding PVE in patients with CIC levels persisting below 30 micrograms/mL.

ループス腎炎の免疫学的研究

Using the Raji cell radioimmunoassay of Theofilopoulos et al, soluble immune complex (IC) in sera from 26 patients with systemic lupus erythematosus was measured. The distribution and amount of renal immune deposits were examined.According to the distribution of glomerular immune deposits, patients were divided into four groups. There were 3 patients with the focal segmental mesangial pattern, 8 with the diffuse global mesangial pattern, 11 with the granular pattern and 4 with the membranous pattern. In 2 of the 3 patients with the focal segmental mesangial pattern, serum IC levels were only slightly elevated and mesangial immune deposits were very light. In 7 of the 8 patients with the diffuse global mesangial pattern, serum IC levels were markedly elevated with large amount of mesangial glomerular immune deposits in spite of minimal urinary finding. In patients with this pattern, wide deviation of serum IC levels was observed. In 7 of the 11 patients with the granular pattern, marked elevation of serum IC levels was observed. Three patients with sclerotic glomerurlar changes had relatively low serum IC levels. In 3 of the 4 patients with the membranous pattern, serum IC levels were low. Of the 26 patients of this study, 12 having renal interstitial deposits had higher serum IC levels than 14 patients without renal interstitial immune deposition. This difference was statistically significant. There were no relationships between the serum IC levels and the grade of hematuria or proteinuria.The data suggests that the serum IC levels measured by the Raji cell radioimmunoassay may give us some information about the degree of renal immune deposition and become a useful index of lupus nephritis.

Circulating immune complexes and their possible relevance to other immunological parameters in Guatemalan onchocerciasis.

Circulating immune complexes (CIC) were demonstrated in sera of Guatemalan patients with onchocerciasis by Raji cell radioimmunoassay. 44% of patients but none of controls had abnormally high concentrations of CIC in their sera. The increased concentrations of CIC were found more frequently in patients with lower density of microfilariae in their skin biopsies. Patients with higher concentrations of CIC appeared to have increased titers of serum antibodies to Onchocerca volvulus. A depression of both humoral immune response to tetanus toxoid and delayed hypersensitivity reaction to PPD were found in patients with onchocerciasis. CIC may be involved in modulation of the immune response in onchocerciasis.

IgA-containing circulating immune complexes in patients with IgA nephropathy

The role of circulating immune complexes in the pathogenesis of IgA nephropathy (Berger's disease) is controversial. Previous studies have shown that a minority of these patients have immune complexes, but the methods used have been able to detect only IgG- or IgM-containing circulating Immune complexes. Using a sensitive, specific Raji cell radioimmunoassay for IgA-containing circulating immune complexes, we have examined serum specimens from 12 patients with IgA nephropathy for the presence of IgA-containing circulating immune complexes. In addition, the Raji cell IgG assay and the 125I-C1q binding assay were used for the detection of IgG-or IgM-containing circulating immune complexes. Purified monoclonal antibodies against human IgA 1 and IgA 2 were used to determine the subclass of IgA present in renal biopsy specimens from five of these patients. Six of 12 (50 percent) patients had IgA-containing circulating immune complexes, whereas only two of 12 (17 percent) had positive results in the Raji IgG assay and one of 12 (8 percent) in the 125I-C1q binding assay. There was no correlation between serum IgA, C3, C4, or factor B levels and the presence or level of IgA-containing circulating immune complexes. None of the three patients with renal failure had circulating immune complexes of any type. Of the seven patients with disease duration of two years or less, five (71 percent) had IgA-containing circulating immune complexes, and three (43 percent) had IgG- or IgM-containing complexes. In all five renal biopsy specimens examined for IgA subclass, diffuse, heavy, mesangial deposits of IgA 1 were seen, whereas IgA 2 staining was absent or present in only trace amounts. These findings suggest that IgA 1 is the predominant antibody in renal biopsy specimens from patients with IgA nephropathy. The finding of IgA-containing circulating immune complexes in these patients—and their more frequent occurrence in patients with early stages of the disease—suggests that IgA-containing circulating immune complexes may play a role in the pathogenesis of IgA nephropathy.

Circulating immune complexes in eosinophilic fasciitis.

Serum immune complexes were measured in 22 patients with eosinophilic fasciitis, 8 of whom had serial determinations. Elevated levels were found by Raji cell radioimmunoassay in 14 (64%) patients, by agarose gel electrophoresis in 13 (59%), and by C1q agglutination-inhibition in 9 (41%). Elevated levels by Raji cell assay were present more frequently at times of active disease [17 of 22 sera (77%)] than at times of inactive disease [2 of 24 (8%) (P less than 0.0005)] and were more closely correlated with disease activity than were eosinophilia, hypergammaglobulinemia, or increased erythrocyte sedimentation rate.

Circulating immune complexes and in vitro cell reactivity in paracoccidioidomycosis.

The severest forms of paracoccidioidomycosis (Pcm) are associated with impaired cell-mediated immunity, a phenomenon that is reversible with therapy. It has been postulated that plasma factors could be responsible for such immune dysfunction. In this report, circulating immune complexes (CIC) were measured by the Raji cell radioimmunoassay (Raji) and by the125I-C1q binding assay (CIq-BA) in sera from 14 patients with either active or inactive forms of Pcm and from 15 healthy controls. The C1q-B A revealed significantly elevated levels of CIC in the sera of all but one of the patients. Four of the 8 active (62%) and 2 of the 6 inactive (33%) patients had CIC levels significantly higher than the controls as determined by the Raji test. Significantly increased levels of CIC were detected only in the active patients by the Raji test. The serum of one of the patients, with a generalized infection and depressed lymphocyte responsiveness, was examined and found to contain a factor which depressed the in vitro proliferation of both homologous and normal lymphocytes. We also found that pre-culture of the patients’ lymphocytes before stimulation restored their proliferative capacity, and IC were detectable in the culture supernatants. However, the subsequent addition of the patients’ serum to such precultured cells did not reinduce the depression. It is suggested therefore, that the depression of T cell responses observed in Pcm is due to the presence of IC which may interact reversibly with the responding cells and/or activate a suppressor cell population whose activity is diminished by preculture.

Immune complexes in primary biliary cirrhosis: Higher prevalence of circulating immune complexes in patients with associated autoimmune features

A longitudinal study, examining the levels of immune complexes serially for three years, in serum from 88 patients with primary biliary cirrhosis was performed by the Raji cell radioimmunoassay. Studies of the association of autoimmune features in primary biliary cirrhosis and the effect of D-penicillamine therapy in relation to the levels of complexes were carried out. Twenty-two patients (25 percent) were found to have autoimmune features, such as Sjögren's syndrome, rheumatoid-like arthritis, scleroderma, Raynaud's disease, and Hashimoto's thyroiditis. In this subset of patients with primary biliary cirrhosis, a significantly higher prevalence (86 percent) of circulating immune complexes was detected compared with those patients showing no autoimmune features (60 percent). In addition, patients with associated autoimmune features had higher mean levels of immune complexes (259.7 μg AHG eq/ml) compared with those without autoimmune features (202.1 μg AHG eq/ml). The mean levels of complement C4, reflecting activation of classic complement pathway, were significantly lower in patients with elevated immune complexes and associated autoimmune features. The mean level of immune complexes in 13 patients receiving D-penicillamine, in contrast to the placebo group, decreased at one year but subsequently was greater than the initial level. Patients who had normal levels of immune complexes and received penicillamine therapy continued to have complex levels within the normal range for up to three years of follow-up study, but patients receiving placebo showed significantly elevated levels at subsequent intervals. Thus, levels of immune complexes in primary biliary cirrhosis may reflect the association with autoimmune features.

Antibody-mediated cellular cytotoxicity against Raji cells ADCC(Raji): evaluation of false positives in the detection of circulating immune complexes by Raji-cell assay.

Raji-cell radioimmunoassay is a very sensitive and reproducible method for the detection of circulating immune complexes. Using a complement-independent, antibody-dependent cellular cytotoxicity assay against 51Cr-labeled Raji cells, there is no correlation between activity against Raji cells and positivity in Raji-cell radioimmunoassay for circulating immune complexes in three sets of sera (from renal transplantation patients, multiparous women, and patients suffering from systemic lupus erythematosus). We conclude that IgG antibodies to Raji membranes are not a significant cause of false-positive results in circulating immune complexes as detected by Raji-cell radioimmunoassay.

The platelet factor 3 assay and circulating immune complexes as studied on non-thrombocytopenic patients with systemic lupus erythematosus.

Sera from 21 patients with systemic lupus erythematosus (SLE) were tested with a platelet factor 3 (PF3) assay. A control serum (pooled blood group AB serum) was always run in parallel with the test sera. The test and control sera were run in duplicate and for each patient the mean recalcification time was used for the calculation of the test serum: AB serum ratio. Concomitantly, the patients' serum concentrations of circulating immune complexes (CIC) were measured by using the Raji cell radioimmunoassay. 20 healthy hospital employees served as controls. In the latter subjects the mean test serum: AB serum ratio was 0.99 (range 0.92-1.09), their CIC concentrations being less than or equal to 16 micrograms/ml. 7 of the SLE patients had CIC values less than or equal to 16 micrograms/ml, and their test serum: AB serum ratios ranged 0.95-1.09. All the remaining SLE patients had elevated values for CIC. In the total material of SLE there was a highly significant (r = -0.87; P less than 0.001) negative correlation between the test serum: AB serum ratios and the values for CIC. It is widely held that the so-called PF3 immunoinjury test detects circulating antiplatelet antibodies. The present results, however, strongly suggest that the assay rather or also detects CIC which like antiplatelet antibodies may damage platelets as we and others have shown in prior publications.

Herpes Simplex Antigen in Immune Complexes of Patients With Erythema Multiforme

Serum samples from patients with erythema multiforme (EM) were examined for the presence of herpes simplex virus (HSV) antigen in immune complexes using the Raji cell radioimmunoassay. Raji-cell-bound immune complexes from eight of 12 patients with EM after HSV infection and four of four patients with EM of uncertain cause had increased anti-HSV binding, while patients with EM after drug exposure and patients with recurrent HSV infections without EM had binding in the same range as controls. Viral cultures for HSV of immune complexes eluted from Raji cells were negative. Sucrose density gradient ultracentrifugation studies of serum samples of patients with EM after HSV infection showed HSV antigen in large molecular weight fractions. The HLA typing of lymphocytes from 16 patients with EM after HSV infection was not different from that of controls. Immune complexes composed of antibody and HSV antigen are present in serum samples of patients with EM after HSV infection and some cases of EM of uncertain cause and may mediate the pathogenesis of these disorders.

Are immune complexes a factor in the pathogenesis of otitis media with effusion?

The authors investigated the presence of IgG-containing immune complexes in middle ear effusions as well as in middle ear mucosa and mastoid mucosa using the Raji cell radioimmunoassay (RIA), the inhibition of anti-antibody, and direct immunofluorescence tests. The Raji cell RIA showed positive results in two of 15 concentrated middle ear effusions but in none of 26 unconcentrated samples. The anti-antibody inhibition assay revealed immune complexes in two of 26 unconcentrated fluids. Thus, with two assays for soluble immune complexes, only four of 63 samples (6 per cent) were positive. Direct immunofluorescence of middle ear mucosa and mastoid mucosa resulted in localization of IgA and IgG in the plasma cells of the middle ear mucosa. A concomitant immunofluorescent staining for complement or immunoglobulin deposits along the basement membrane of the epithelium and/or blood vessels was not observed in 37 biopsy specimens. These findings do not support the concept that immune complexes are involved in the pathogenesis of otitis media with effusion.

Circulating immune complexes during various forms of renal allograft rejection episodes.

Previous reports on the nephritogenic and immunological enhancing capacity of posttransplant circulating immune complexes (CICs) are conflicting and have been confined to the study of acute rejection episode (AR). This study was undertaken to assess the nephritogenicity of CICs in patients undergoing accelerated acute rejection (AAR) and chronic rejection (CR) episodes. We also assessed the possible role of CICs as mediators of immunological enhancement by assessing CIC levels in long-term-stable allograft recipients. To asses the nephritiogenic role of CICs, 98 CIC determinations were performed on 49 serum samples from 41 pediatric renal transplant recipients using the C1q solid-phase assay (C1q-SPA) and the Raji cell radioimmunoassay (Raji-RIA). Serum samples from normal subjects served as controls. No recipient undergoing AAR had evidence of CICs by eigher assay. 5 of 21 (23.8%) recipients undergoing CR had positive CIC levels in the Raji-RIA, while 4 of 21 (19%) recipients had positive CICs inthe C1q-SPA. There was no statistically significant correlation of CIC levels with long-term allograft function. In addition, there was no evidence supporting antithymocyte globulin as an immunogen in patients demonstrating posttransplant CICs. In summary, CICs do not appear to be an important mediator of AAR or CR episodes, and CICs were not routinely detected in patients with good long-term allograft function.

Circulating immune complexes in rheumatic fever.

We measured the circulating immune complex levels in patients with rheumatic fever using the following three methods: the C1q binding test, the conglutinin binding test and Raji cell radioimmunoassay. Thirty-four samples from 21 patients with rheumatic fever were examined, and circulating immune complexes were detected in 47.6% of the patients by one or more of these methods. Especially by the C1q binding test, the sera obtained in the acute phase were found to contain the circulating immune complexes with higher frequency than those obtained in the post-acute phase. Therefore, the C1q binding test was more likely to be useful in the acute phase of this disease. The present study suggests that the circulating immune complexes are related to the pathogenesis of rheumatic fever.

Abnormalities of pulmonary vascular dynamics and inflammation in early progressive systemic sclerosis.

Abnormalities of pulmonary function were studied in 10 patients with progressive systemic sclerosis (PSS) and 3 control subjects. All underwent 81M krypton lung scanning and total body gallium scanning. Immune complexes were measured by Raji cell radioimmunoassay and polyethylene glycol (PEG) assay. Perfusion scans were abnormal in 7 of 9 patients, and 5 of 9 showed a decrease in pulmonary perfusion after cold challenge. Increased gallium uptake was noted in the lungs of 6 of 9 patients. Krypton scans were normal in the control group. Elevated immune complexes were noted in 8 of 10 patients by the Raji assay and in 5 of 10 with the PEG assay. Efforts to separate patients with PSS into subgroups may lead to a better understanding of and advances in therapy for PSS.

Evidence that circulating immune complexes remove transfused platelets from the circulation.

Fifteen recently diagnosed patients with acute leukemias admitted for induction chemotherapy were selected for study. When thrombocytopenic (venous platelet count less than 20 X 10(9)/l) these patients received prophylactic platelet transfusions. A total of 67 platelet transfusion therapies were administered and evaluated. Using the Raji cell radioimmunoassay, the serum concentrations of circulating immune complexes (CIC) were measured immediately before and 10-12 hr after each platelet transfusion. In 36 instances, elevated values of CIC were present in the recipient's pretransfusion samples. The corresponding posttransfusion values being significantly lower (P less than 0.05). Furthermore, in those 36 instances the mean percentage for the posttransfusion platelet increment was significantly lower (P less than 0.001) than in the remaining 31 instances in which normal pretransfusion values of CIC were measured. We conclude that CIC were an important factor in rapidly removing transfused platelets from the circulation, thereby, adversely affecting the benefit of platelet transfusions.

Immune abnormalities in IgA nephropathy (Berger's disease)

Thirteen patients with IgA nephropathy confirmed by renal biopsies were studied for evidence of disorders of the immune system. Circulating immune complexes (CIC) were determined by agarose gel zone electrophoresis (AGE), the Raji cell radioimmunoassay (RIA), and the modified Raji cell (RIA) for IgA containing CIC. Serum levels of immunoglobulins (Ig) G, A, M, of C3, C4, and properdin factor B (PFB) were measured by the rate nephelometry. Tissue studies included fluorescent, light, and electron microscopy, staining for intracellular Ig, and lysozyme by immunoperoxidase (IP), and determination of glomerular C3 receptors (C3R) by EAC rosettes. IgG-CIC were detected in 69%, IgA-CIC in 50% and serum IgA was elevated (>2SD) in 46% of the patients. In the glomeruli, mesangial deposits of IgA, IgG, properdin, and C3 were present. C3R available for EAC binding to glomerular cells were decreased. IgA-producing lymphoid cells were detected in renal interstitium of our patients especially those with crescentic glomerulonephritis. Elevated serum IgA and the presence of IgA-producing cells in the renal tissue may reflect an abnormality in IgA immunoregulatory mechanisms. IgA could contribute to the pathogenesis of renal disease due to its inhibitory effect on the clearance of circulating and deposited immune complexes.

CIRCULATING IMMUNE COMPLEXES IN PEDIATRIC RENAL ALLOGRAFT REJECTION

Previous reports on the generation and nephritogenic capacity of post-transplant circulating immune complexes (CICs) are conflicting. To assess the pathogenicity of CICs in acute rejection (AR), 784 CIC determinations were performed on 392 serum samples from 27 pediatric renal allograft recipients using the C1q-solid phase assay (C1q-SPA) and the Raji cell radioimmunoassay (Raji-RIA). Serum samples from transplant recipients not undergoing rejection episodes and from normal subjects served as controls. Of the 784 CIC determinations, 723 (92.3%) were negative in both assays. CICs were present at some point post-transplant in eight (19.6%) recipients. Correlation of CIC levels with allograft rejection was found in only two patients with CIC levels responding to antirejection therapy; however, statistical analysis of data by chi 2 analysis failed to reveal a significant correlation of CICs with AR episodes. Allograft histology in three recipients demonstrated characteristic signs of AR. Immunofluorescent studies did not reveal significant deposition of immunoglobulin or complement. Sucrose density gradient ultracentrifugation studies confirmed the immune complex nature of materials reactive with the CIC assays. There was no immunological evidence supporting antithymocyte globulin (ATG) as an immunogen in patients demonstrating CICs post-transplant. CICs do not appear to be an important mediator of AR. Statistical analysis of data using the chi 2 test failed to reveal a positive correlation of CIC levels with AR or ultimate allograft outcome.