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In a collaborative study involving three laboratories, randomly coded sera from 47 patients and healthy donors were tested for soluble immune complexes by two versions of the C1q BT and by the C1q DT. Analysis of ranked data showed a close correlation between results obtained in all laboratories as well as good reproducibility on testing coded duplicate samples included in each panel of sera. However, with the use of values for normal donors established independently in each laboratory, the tests did not always agree in discriminating normal from abnormal sera, particularly with sera from cancer patients. Results reflected to some extent the methods used to inactivate the endogenous C1 complex in the test sera. Studies of 130 additional cancer patients revealed that 44 (34%) gave abnormal C1q BT values when inactivated with EDTA but only 35 (27%) were abnormal when heat inactivated (56 degrees for 30 min). Similarly 12 of 65 sera (18%) from patients with nonneoplastic diseases and 10 of 80 (13%) from healthy donors gave significantly abnormal results after addition of EDTA. Eight (12%) of the disease and six (8%) of the healthy controls' sera were similarly outside normal limits when heat inactivated. Repeated freezing and thawing of sera or changes in the concentration of PEG used to precipitate complex-bound 125I-C1q influenced C1q BT results more than duration of storage at -70 degrees C or changes ascribed to presence or absence of rheumatoid factors and CRP.

A comprehensive immunologic and serologic analysis was performed on 31 untreated patients with Hodgkin's disease. Immune evaluations stressed T-cell functional activity and included traditional parameters (PHA responsiveness and delayed hypersensitivity skin reactivity), as well as newer functional assays (T-cell colony formation, chemotaxis, spontaneous and antibody-dependent cytotoxicity, and concanavalin A-induced suppressor cell activity (CISA). Serum factors included ferritin, prostaglandins, zinc, copper, immune complexes, and thymic hormone activity. Every patient exhibited at least one T-cell or serum abnormality. The greatest percentage of patients exhibited T-cell defects in chemotaxis (85%), colony formation (81%). and PHA reactivity (64%). Immune defects were more common with advanced disease but were not related to absolute T-cell or monocyte count, skin test anergy, or abnormalities of T mu/T gamma cell proportions. Linear relationships were identified among abnormalities in the three assays employing mononuclear cells (PHA, colony formation, CISA) which may have reflected the inhibitory influence of monocytes present in the mononuclear cell preparations. Low serum zinc correlated with marked impairment of T-cell chemotaxis. Elevated prostaglandins were associated with high PHA reactivity and with depressed colony formation. Our results indicate that many complex factors, including intrinsic T-cell defects, contribute to the impaired immunity associated with Hodgkin's disease.

To test the possibility that autoimmunity could be related to increased levels of anti-bovine antibodies and/or circulating immune complexes in selective IgA deficiency, we studied the sera of 30 consecutive patients for the quantitative level of antibody to bovine milk, the presence of antigen--antibody complexes and 10 selected autoantibodies. Higher titres of anti-milk antibody and circulating immune complexes were to be correlated with positive serological tests of autoimmunity in these patients, and rheumatoid arthritis (three cases) and neurological disease (four cases) were found in individuals who had both milk precipitins and circulating immune complexes. We suggest that the chronic excessive permeability of the gastrointestinal tract in selective IgA deficiency may permit the excessive absorption of many food proteins, leading to the formation of antigen--antibody complexes and autoimmunity.

Sera of normal individuals or patients with lung cancer were assayed for Forssman-like antibody by a quantitative and specific method using ethylenediaminetetraacetate-containing buffer to inactivate complement in the test serum. It was shown that although Forssman-like antibody levels were distributed widely, (a) the levels of young (20 to 45 years of age) normal subjects of Blood Groups A and AB were lower than those of Blood Groups O and B, (b) the levels of old (60 to 80 years of age) normal subjects were lower than those of young normal subjects of Blood Groups O and B, and (c) the levels of old lung cancer patients were lower when compared to age-matched normal individuals of their blood group.

The presence of Fc receptors, but not C3 receptors, on human and bovine spermatozoa was shown by sperm-induced rosette formation of antibody-treated ox erythrocytes and by immunofluorescence of aggregated human gammaglobulin (AHG) treated spermatozoa. Human and bovine spermatozoa bound AHG as well as circulating immune complexes (CIC) from human sera; monomeric IgG bound to these cells to a much lesser extent. Binding of AHG to the sperm surface also occurred when the AHG was suspended in either buffer or in serum that had been heated at 56 degrees C for 30 min. A bovine spermatozoa radioimmunoassay for the detection of immune complexes in human sera was developed and was shown to be comparable to the Raji cell radioimmunoassay (r = 0 . 81, P < 0 . 01) in the quantitation of soluble immune complexes from a variety of human sera. An enzyme-linked immunoassay was then developed, using bovine spermatozoa fixed to wells of microtitre plates, to simplify the immune complex detection assay. The technique provides an analysis that is rapid, does not require radioactive chemicals, expensive equipment, or cultured cells and yields values for immune complex determination both comparable and complementary to the Raji cell radioimmunoassay (r = 0 . 70, P < 0 . 01). The sperm assays detected soluble immune complexes in sera of vasectomized males, cancer patients and patients with autoimmune or dermatological diseases.

IgG and circulating IgG immune complexes (CIC) were purified from plasma of three pet cats persistently infected with feline leukemia virus (FeLV) by adsorption to, and elution from, Staphylococcus aureus Cowan I. CIC were then separated from free IgG by sucrose gradient ultracentrifugation and were analyzed for the presence of FeLV structural proteins and corresponding specific antibodies. Radioimmunoprecipitation analysis indicated that FeLV envelope (gp70) and major core (p30) proteins, along with cat IgG heavy and light chains, were present in the CIC from all three cats. Further analysis of the CIC from one of the cats also revealed the presence of FeLV core proteins p15 and p12. IgG purified from isolated CIC was also shown to bind specifically to purified FeLV gp70, p30, and p15. These data provide direct evidence for FeLV-specific CIC in the plasma of persistently viremic pet cats, and suggest these animals are immunologically response to the virus even though free antibodies against the major structural proteins cannot be demonstrated in standard assays.

Cats were classified into 4 categories by immunofluorescence antibody assay for the presence of feline leukemia virus (FeLV) and histologically as a) normal, FeLV-, b) normal, FeLV+, c) lymphosarcoma (LSA), FeLV+, and d) LSA, FeLV-. Determinations by Raji cell radioimmunoassay modified for cat serum revealed circulating immune complex (CIC) levels of healthy cats to be less than or equal to 50 micrograms equivalent aggregated cat immunoglobulin/ml (microgram/ml). In contrast, sera of cats in groups b, c, and d all contained significantly higher CIC levels (up to 12,000 micrograms/ml) associated with marked hypocomplementemia and C activation occurring via the classical pathway. Sera from FeLV+, LSA+ cats with high levels of CIC and sera of healthy cats were fractionated according to size and bouyant density by centrifugation through 10 to 40% sucrose gradients. Analysis of fractions from LSA+, FeLV+ sera revealed that both immune complexes (ICs), FeLV reverse transcriptase (RT), and IgG were present in fractions corresponding to a bouyant density of 1.15 to 1.18 g/ml. The CIC containing fractions activated C by the classical pathway. Sera from normal cats did not have CIC or RT and none of the fractions activated the classical pathway. These data suggest that vital antigen-antibody complexes are present in sera of viremic cats with LSA and these complexes activate the C system.

E, pretreated with leukocyte cultures supernatant (ES), binds C1 through C1q; ES and EIgM that bind the same amount of C1 as measured in a hemolytic assay have the same uptake of 125I-C1q; ESC1q and EIgMC1q, carrying the same number of molecules of CUq per cell, have the same uptake of CUr and CUs; soluble immune compleses prevent the binding of C1 and C1q to ES. The activity of C1 bound to ES is impaired; ESC1 can react with C4 but not with C2. The C4 turnover and the C1 ING turnover by ESC1 are reduced so that ES-bound C1 is protected from destruction by C1 ING. These modifications are fully reversed when C1 is transferred from ES to EA:C1 recovers its ability to react with C2, and C1 INH. Thus the C1s activity can be modulated inside the C1 molecular complex upon binding of C1q to a lymphocyte product. In addition, the 125I-C1q uptake is proportional to the amount of IgM hemolysin used to sensitize E; it has, however, an exponential relationship to the amount of IgG or S used to sensitize E. The ratio of 125I-C1q uptake towhole C1 uptake measured in a hemolytic assay is lowerthan 2. This indicates that one molecule of IgM is sufficient to bind one molecule of C1q on E, that several molecules of IgG or S are required to bind one molecule of C1q, and that one molecule of C1q is sufficient to create a lytic site on E.

Unmeasurable total haemolytic complement (C) was observed in serum of a patient with untreated chronic lymphocytic leukaemia and recurrent non-hereditary angioedema. Analysis of C components immunochemically demonstrated a marked reduction of C1q and C1s inhibitor, undetectable C1r, C1s and an elevated B. Haemolytic C1, C4 and C2 were less than 5 percent of normal, functional C1s inhibitor was absent. Cryoglobulin and C1q precipitins were present in the serum. Of special interest was the presence of high levels of cold-reactive antilymphocyte antibody, determined by both C-dependent cytotoxicity and indirect immunofluorescence. The antibody exhibited specificities for both autologous lymphocytes and lymphocytes from normal donors; cytotoxic activity for autologous leukaemia cells was removed by absorption with normal isologous tonsil lymphocytes. Specific enrichment of this antibody relative to the serum level was demonstrated in the cryoglobulin and its isolated 19S fractions. Free lymphocyte surface antigen was also demonstrated by gel diffusion using specific rabbit antilymphocyte antiserum. These data strongly suggest the presence of pathogenetically significant circulating complexes of lymphocyte surface antigen and specific antibody in certain patients with CLL.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of immune complexes in sera has been developed using Raji cells. In this assay the Raji cell-adherent complexes are detected by an alkaline phosphatase-conjugated rabbit anti-human IgG and the results are obtained by spectrophotometric measurements of the concentration of yellow colour produced after the enzyme's substrate, p-nitrophenyl phosphate, is added. This assay is as sensitive and reproducible as the Raji cell radioimmune assay using 125I, as it can detect as little as 16 micrograms of immune complex equivalent to heat-aggregated IgG/ml in serum samples. This method is inexpensive, convenient and most importantly, avoids the biohazards of using an isotope.

Eighteen patients with agnogenic myeloid metaplasia with myelofibrosis were studied for clinical and laboratory evidence of immunologic dysfunction. Clinical findings included the presence of arthritis, vasculitis, and erythema nodosum. Laboratory abnormalities included the presence of circulating immune complexes, antinuclear antibodies, positive direct Coombs tests, elevated latex fixations, and a circulating lupus type anticoagulant. Total hemolytic complement was markedly depressed in four patients. Analysis of complement (C) components C1-C9 and factor B demonstrated significant reduction of only C3 and factor B. By crossed-immunoelectrophoresis, both C3 and factor B, but not C4, were cleaved, indicating that C activation was occurring predominantly via the alternative pathway. The control proteins beta 1H and C3b inactivator were decreased in three of four patients with hypocomplementemia. These data suggest that immunologic mechanisms associated with activation of the complement system play an important role in the disease process of some patients with agnogenic myeloid metaplasia with myelofibrosis.

Serum levels of circulating immune complexes (CIC) assayed by the Raji cell radioimmunoassay, total haemolytic complement (TCH50), Clq and C3 were correlated with clinical stage, histological type, age, sex and treatment of eighty-six children with Hodgkin's disease over a period of 4 years. Most significant findings were the changes of levels of CIC, TCH50, Clq and C3 during disease activity and following treatment. Significant perturbations were also seen in association with relapse. Levels of C and CIC were significantly elevated (P less than 0.001) at the time of diagnosis prior to splenectomy and/or any treatment. In the group before treatment, 81 percent of CIC levels were above 16 micrograms/ml with a maximum value of 1120 micrograms/ml. During treatment 33 percent were still above normal with a maximum of 320 micrograms/ml. Within 1 year after cessation of treatment, 37 percent also remained above normal levels with a maximum of 240 micrograms/ml. At relapse prior to treatment, 63 percent were again elevated with a maximum of 1280 micrograms/ml. The most significant difference on TCH50 levels relates to treatment periods. Sera of patients with active disease who are previously untreated show elevation of TCH50 levels (P less than 0.001) (average 127 CH50 mu/ml. During and after treatment eht TCH50 levels drop to 96 and 102 CH50 mu/ml, as compared to normal control of 100 CH50 mu/ml. In sera of patients at the first, second or third relapse, the combined TCH50 levels are significantly different from controls and across treatment periods (P less than 0.005).

A 44 year old woman with common variable immunodeficiency developed severe anaphylactic reactions to intravenous gammaglobulin. Analysis of her serum prior to the infusion of gammaglobulin was thus analyzed and the tests revealed a complete absence of free IgA, presence of an IgG autoantibody to IgA, IgA-IgG circulating immune complexes, and depressed levels of hemolytic C3 associated with elevated levels of C3a and C3d. The IgA-IgG complexes did not clear from the circulation even after six months following cessation of gammaglobulin infusions. Analysis of the complexes isolated by sucrose density gradient ultracentrifugation showed that they bind to solid phase F(ab')2 anti-C1q, have a high molecular weight (greater than 19s), activate the complement (C) system via the classical pathway in vitro and are comprised of IgG and IgA. These data suggest that in this patient an autoantibody response to IgA was probably associated with persistent endogenous production of IgA yielding IgG-IgA circulating immune complexes and activation of the complement system. Although the patient has no free IgA and no surface IgA bearing B cells, her peripheral blood lymphocytes were shown to contain cells capable of secreting IgA. Low levels of IgM and IgG were detectable in her serum, and B cells bearing surface IgM, IgG and IgD were present in normal numbers in the blood.

Levels of circulating immune complexes were determined in the Trinidadian population with a high incidence of acute post-streptococcal sequelae, acute rheumatic fever and acute post-streptococcal glomerulonephritis. The studies were done on acute phase and serial bleedings up to 6 months after the acute illness. C1q solid phase assay employing radiolabelled protein A and the Raji cell radioimmunoassay were used in the detection of immune complexes. Both groups of patients had significantly elevated levels of immune complexes during the acute phase and showed an equivalent decline to slightly elevated levels during the 6 months of observation. The incidence and molecular size of the immune complexes in the two post-streptococcal sequelae were equivalent. The Raji cell assay tended to show much higher levels of complexes on an absolute basis, but both assays demonstrated an equivalent decline from the acute phase. These studies suggest that the presence of equivalent levels of complexes in two entirely different post-streptococcal sequelae indicates that other factors, such as the nature of the antigen in the complex, are more important in the pathogenesis of the post-streptococcal sequelae than the actual presence or the absolute levels of complexes.