Circulating immune complexes in coccidioidomycosis. Detection and characterization.

Sera of 22 patients with active and 13 with inactive coccidioidomycosis, as well as 15 healthy subjects who were skin-test positive to coccidioidin and 39 healthy subjects who were coccidioidin skin-test negative, were assayed for immune complexes. Circulating immune complexes were measured by the Clq-binding assay, the Clq-solid phase assay, the monoclonal rheumatoid factor inhibition assay, and the monoclonal rheumatoid factor solid phase assay. An increased concentration of circulating immune complexes was detected in 73% of those with active disease by at least one assay compared with 13% of the healthy controls. Significantly increased levels of immune complexes were detected in sera of patients with active coccidioidomycosis by the Clq-binding assay (P < 0.001), the Clq-solid phase assay (P < 0.001), the monoclonal rheumatoid factor inhibition assay (P < 0.005), and the monoclonal rheumatoid solid phase assay (P < 0.05) compared with the results obtained in the 54 healthy subjects. In contrast, those with inactive disease did not show significantly increased concentrations of circulating immune complexes. Sucrose density gradient ultracentrifugation of patients' sera established that the immune complexes were of intermediate size, sedimenting between the 6.6S and 19S markers. Immune complexes were shown to contain both coccidioidin antigen and anticoccidioidin antibody. In addition, a radioimmunoassay was developed to quantitate coccidioidin antigen-containing immune complexes. The latter assay proved highly sensitive in detecting immune complexes in patients with active coccidioidomycosis.     

Evaluation of the C1q solid-phase binding assay for immune complexes. A clinical and laboratory study.

The solid-phase C1q binding assay for circulating immune complexes has been evaluated. The assay provides a rapid, sensitive (detecting as little as 1 microgram of aggregated IgG) and reproducible procedure for the detection of immune complexes in biological fluids. Using artificially prepared immune complexes, the assay detects complexes at four-times antigen-excess. Gel filtration over Sepharose 6B showed that these complexes were distributed over a range of molecular weights from greater than 4 x 10(6) to 300,000 daltons. Using radiolabelled anti-BSA, antigen (BSA) could be detected in these complexes. Screening of gel-filtered SLE showed that the assay detects complexes of both high and low molecular weight, but does not detect all complexes in the SLE sera. Clinical studies showed that immune complexes are frequently found in the sera of patients with SLE and measurement of the concentrations of complexes provides a more sensitive index of disease activity than either serum C3 or C4 concentrations or DNA binding capacity. In patients with RA concentrations of immune complexes were generally higher in synovial fluid than serum, although a patient with systemic rheumatoid disease with hypocomplementaemia had an extremely high level of circulating immune complexes. The assay only infrequently detects circulating immune complexes in glomerulonephritis and in renal transplant recipients. It is concluded that the assay provides a useful clinical tool, but detects only a limited species of immune complexes. It can be used in the detection of antigens in complexes.     

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [(125)I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 mug and that of soluble human IgG-anti-IgG complexes is about 3 mug of complexed antibody. Some immune complexes formed in large antigen excess (Ag(2)Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab')(2) antibody complexes to lead to a precipitation of [(125)I]C1q in PEG.In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes.Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [(125)I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients.No increased [(125)I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [(125)I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [(125)I]C1q. The results were also used for a correlative study of [(125)I]C1q binding to IgG levels in the sera but increased [(125)I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates.These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.     

Immune complexes in sera and synovial fluids of patients with rheumatoid arthritis. Radioimmunoassay with monocylonal rheumatoid factor.

Evidence for the presence of immune complexes in blood, synovial fluid, and tisues of patients with rheumatoid arthritis (RA) includes low complement levels in blood and effusions, deposition of immunoreactants in tissues and vessel walls, precipitate formation after addition of monoclonal rheumatoid factor (mRF) to serum or synovial fluid. To quantitate immune complex-like material in RA patients, we developed a radioimmunoassay based on inhibition by test samples of the interaction of (125I)aggregated IgG (agg IgG) and mRF coupled to cellulose. This method could measure immune complexes of human antibody with hemocyanine prepared in vitro. The assay was not influenced by presence of polyclonal RF in test samples, nor by freezing and thawing. Normal levels of immune complex-like material in serum were less than 25 mug agg IgG EQ/ML. 12 of 51 RA sera examined (26%) contained more than 25 mug/ml. The presence of this material in RA sera was found to correlate with severity of disease, as measured by anatomical stage and functional class. There was an inverse correlation of the material with serum C4 level. Rheumatoid synovial fluids generally contained higher levels than serum, and five of 23 contained very much higher levels. The frequency of elevated levels of immune complex-like material in sera of patients with systemic lupus erythematosus (2 of 29) and with miscellaneous vasculitides (2 of 21 was much lower than in RA, suggesting that mRF exhibits a specificity for only certain kinds of immune complexes. The reason for this apparent specificity may explain such distinctive features of RA as the high frequency of polyclonal RF, the lack of immune complex nephritis, and the generally normal levels of serum complement.     

Inhibition of polymorphonuclear leukocyte chemiluminescence for detection of immune complexes in human sera.

An assay for the detection and quantitation of immune complexes is described. Experimental immune complexes or aggregated human gamma globulin (AHG) were incubated with polymorphonuclear leukocytes (PMN). After challenge of the PMN with opsonized zymosan, chemiluminescence was recorded in a scintillation spectrometer. A quantitative inhibition of chemiluminescence could be demonstrated by the interaction of PMN with immune complexes or AHG. Experimental immune complexes of bovine serum albumin-anti-bovine serum albumin were formed and tested by this assay, and immune complexes formed near antigen excess were best described by this technique. The technique was used to demonstrate immune complexes in the sera from patients with systemic lupus erythematosus, rheumatoid arthritis, and vasculitis. Immune complexes were quantitated by reference to a standard curve using AHG. By this technique, normal human sera had < 10 micrograms of AHG per milliliter of serum. Immune complexes at levels above this were detected in 9/15 patients with systemic lupus erythematosus, 18/30 patients with rheumatoid arthritis, and 2/5 patients with vasculitis. Therefore, this assay is a sensitive, simple method for measurement of circulating immune complexes in the sera of patients with certain connective tissue diseases.     

Circulating immune complexes in patients with rheumatoid arthritis and the effect of penicillamine in vitro

Protein precipitated from the sera of 15 patients with rheumatoid arthritis (RA) by increasing concentrations of polyethylene glycol 6000 (PEG) was compared with that from 20 normal controls. The wide range of precipitable protein obtained was only significantly different for the two groups when a 4% PEG concentration was used. To increase the discrimination of the procedure, the precipitated protein was measured for IgG, IgM, C3 and C4 using immunospecific antisera and laser nephelometry. The data obtained from each of these procedures did not increase the specificity of the technique as a means of distinguishing between patients and controls. As part of an investigation of the effect of penicillamine on immune complexes, penicillamine was incubated at 37 degrees C for 24 and 48 h with sera containing immune complexes from RA patients. When the immune complexes were precipitated with PEG, it was found that incubation, by itself, reduced the amount of PEG precipitable protein. The results of in vitro incubation with penicillamine were very variable.     

Binding of complement component C5 to model immune complexes and the use of anti-C5 antibodies for determination of C5-containing circulating immune complexes

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.     

Circulating Immune Complexes in Lyme Arthritis: DETECTION BY THE 125I-C1q BINDING, C1q SOLID PHASE, AND RAJI CELL ASSAYS

We have found immunoglobulin (Ig) G-containing material consistent with immune complexes in the sera of patients with Lyme arthritis. It was detected in 29 of 55 sera (55%) from 31 patients by at least one of three assays: (125)I-C1q binding, C1q solid phase, or Raji cell. The presence of reactive material correlated with clinical aspects of disease activity; it was found early in the illness, was most prominent in sera from the sickest patients, was infrequent during remissions, and often fluctuated in parallel with changes in clinical status. The results in the two C1q assays showed a strong positive correlation (P<0.001). They were each elevated in 45% of the sera and were usually concordant (85%). In contrast, the Raji cell assay was less frequently positive and often discordant with the C1q assays. In sucrose density gradients, putative circulating immune complexes sedimented near 19S; they, too, were detected best by the two assays based on C1q binding. An additional 7S component was found in some sera by the (125)I-C1q binding assay. Serum complement was often above the range of normal in patients with mild disease and normal in patients with severe disease but did not correlate significantly with levels of circulating immune complexes. IgM and IgG rheumatoid factors were not detectable.These findings support a role for immune complexes in the pathogenesis of Lyme arthritis. Their measurement, by either the (125)I-C1q binding assay or by the C1q solid phase assay, often provides a sensitive index of disease activity. Moreover, the complexes are likely sources of disease-related antigens for further study of this new disorder.     

A C1q solid phase microenzymatic assay for the detection of soluble immune complexes

Summary The solid phase C1q-binding assay has been adapted to an enzymatic micromethod in which alkaline phosphatase labeled solubleStaphylococcus aureus protein A is used in place of the second antibody. The assay, which is run in microtiter plates, provides a rapid, sensitive (0.030 mg/ml of human heat-aggregated IgG detected) and reproducible method for the measurement of soluble immune complexes in a large number of samples. Soluble immune complexes preparedin vitro with bovine serum albumin (BSA) and anti-BSA antibodies on a wide range of antigen to antibody ratios were all detected with this method. When applied to the screening of unselected patient sera, soluble immune complexes were frequently found in systemic lupus erythematosus (52%) and chronic active hepatitis (57%) and in lower percentages in patients with malignant melanoma (28%), rheumatoid arthritis (30%) and essential mixed cryoglobulinemia (17%). This study was supported in part byConsiglio Nazionale delle Ricerche (CNR), Roma, Italy, ‘Progetto Finalizzato Controllo della Crescita Neoplastica’, grant no. 80.01599.96.     

Raji cell assay for immune complexes. Evidence for detection of Raji-directed immunoglobulin G antibody in sera from patients with systemic lupus erythematosus.

We asked whether binding of human immunoglobulin (Ig)G antibody reacting with Raji cells could be distinguished from binding of IgG immune complexes. Using a standard Raji assay employing 125I-IgG goat anti-human Fc gamma, we found that digestion of Raji cells with pronase reduced by 95% their ability to bind complement-fixed aggregated human gamma globulin and complement-fixed tetanus toxoid-antitetanus toxin complexes. However, binding at 37 degrees C of IgG from the sera of 16 patients with systemic lupus erythematosus (SLE) to pronase-digested Raji cells was reduced much less consistently and extensively (9-100% reduction; mean reduction of 51%). In more detailed studies of two SLE sera, sucrose density gradient centrifugation showed that greater than 50% of the IgG binding to undigested Raji cells sedimented in the 7S region. Pepsin digestion of immunoglobulin fractions from four SLE sera caused a reduction in SLE IgG binding to undigested Raji cells when detected with 125I anti-Fc gamma, but an increase when binding was detected with 125I-anti-Fab, suggesting that substantial SLE IgG can bind through F(ab')2 regions. Binding of IgG from SLE sera was not directed at neoantigenic sites induced by pronase digestion because binding activity was adsorbed with undigested cells as readily as with digested cells. Moreover, sera from 10 SLE patients that had negative Raji assays contained no IgG that bound to pronase-digested Raji cells. We conclude that much of the IgG bound at 37 degrees C to Raji cells from the sera of many patients with SLE does not represent immune complexes but is probably antibody directed toward sites on the Raji cell.     

Effect of circulating immune complexes on transfusional therapy in patients with hemophilia or von Willebrand's disease

Circulating immune complexes were examined in patients with hemophilia or von Willebrand's disease in order to determine the immediate or long- term side effects after transfusion. The conglutinin binding assay which allows quantitation of C3bi-bearing immune complexes was used for 82 patients with hemophilia A. Immune complexes were detected in 37 (45%) of these cases prior to transfusion. Immune complexes also were detected in four of 11 patients with hemophilia A and factor VIII inhibitors, in five of 11 patients with hemophilia B, and in three of 10 patients with von Willebrand's disease. The levels of circulating immune complexes in 21 patients with hemophilia A and seven with von Willebrand's disease significantly increased 24 hours after concentrate or cryoprecipitate transfusions. Purified immune complexes from three patients with hemophilia A were shown to contain IgG, IgM, and complement components. No factor VIII coagulant or antigenic protein or fibrinogen was identified in the immune complexes using specific antisera. Side effects immediately after transfusion were not associated with immune complexes. The levels of factor VIII or IX after transfusion were not particularly decreased in relation to the presence of immune complexes. Finally, the presence of circulating immune complexes in the patients studied did not correlate with the number of transfusions, the units of concentrates injected, the presence of HBsAg or HbsAb, the levels of plasma aspartate transferase, or the presence of rheumatoid factor. Proteinuria was absent in all the patients studied.     

Estimation of immune complexes by a microplate-adapted C1q-Protein A enzyme-linked-immunosorbent-assay (C1q-PA-ELISA)

A microplate-adapted enzyme linked immunosorbent assay (ELISA) for detection of C1q-binding immune complexes (IC) and aggregated IgG (delta IgG) is described. Purified human C1q was adsorbed to the wells of flat-bottomed microtiter plates and EDTA-treated serum samples were subsequently introduced. Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels of IC were found in the majority of sera from patients with rheumatoid arthritis and SLE. The described C1q-PA-ELISA is a simple and inexpensive method for detection of C1q-binding immune complexes. The reproducibility is acceptable and the sensitivity is higher than for most IC-methods based on C1q-binding.     

Cryoimmunoglobulinemia in rheumatoid arthritis. Significance in serum of patients with rheumatoid vasculitis.

Cryogloculins were examined in a standardized manner in an unselected group of 35 patients with rheumatoid arthritis (RA) and 8 patients with RA complicated by cutaneous vasuclitis and neuropathy. Optimum conditions for detection and characterization of cryoglobulins were established; the proportion of resolubilized to total precipitable protein remained constant in an individual patient under these conditions. All 8 vascultis patients and 9 of 35 other patients with RA exhibited cryoglobulins; total protein and immunoglobin content were significantly higher in the cryoglobulins of patients with vasculitis. Immunoglobulins G and M constituted two-thirds and three-quarters of the total protein in the cryoglobulins from uncomplicated rheumatoid and vasculitis patients, respectively. Serum antiglobulin titers were higher, and serum C3 levels were lower, in vasculitis patients compared to rheumatoid patients without vasclitis. Anti-gamma globulin activity was detected in all cryoglobulins from vasculitis patients. Cryoglobulin IgG and IgM were polyclonal. Density gradient analyses demonstrated the majority of the cryoglobulin activity to reside in the 19S IgM fraction. There was no evidence of a light weight (8S) IgM. A monoclonal rheumatoid factor did not detect 7S-ANTI-7S complexes in the cryoprecipitates, but acid eluates from some cryoglobulins absorbed with insoluble IgG revealed an antiglobulin of the IgG class. Serial studies performed on vasculitis patients treated with cyclophosphamide disclosed a relationship between clinical evidence of vasculitis and the presence of cryoglobulins. The antigen (IgG) and antibody (largely IgM rheumatoid factor) nature of these cryglobulins is presented as evidence that the widespread vascular complications of RA are mediated, at least in part, by circulating immune complexes.     

Detection of immune complexes. The use of radioimmunoassays with Clq and monoclonal rheumatoid factor.

This study describes two sensitive, rapid, relatively simple, competitive inhibition radioimmunoassays for detecting immune complex. The tests are based on the inhibition of I125-Clq or I125-monoclonal rheumatoid factor (mRF) binding to an insoluble substrate, IgG-Sepharose. The assays can be performed in 5 h utilizing 10 micronl of serum. Heating of serum is not required and polyclonal rheumatoid factors do not interefere. With the two assays, a wide range of complexes of various size and complement fixing activity can be detected. The Clq test can detect complement fixing Ig complexes larger than 19S, while the mRF tests detect complexes of IgG as small as 8S irrespective of their complement fixing activity. Mouse, rabbit, and human aggregated IgG (agg IgG) can be detected in the Clq test, and human and rabbit agg IgG in the mRF test. As low as 4 microng/ml of isolated human agg IgG can be detected in the Clq test and 0.5 microng/ml in the rheumatoid factor test. Sensitivity is greater for mouse agg IgG. For pathologic sera which must be diluted to eliminate interfering factors, the sensitivity of the assay is approximately 10 times less. The Clq test showed marked inhibition by systemic lupus erythematosus sera with close correlation with CH50 levels and disease activity. The mRF test showed better correlation with rheumatoid arthritis sera. In addition, anionic macromolecules known to react with Clq and other Clq reactants that occur in pathologic sera such as the "low molecular weight" substances in systemic lupus erythematosus are also detected. These reactants are not detectable in the mRF test and can be eliminated in the Clq test by performing the test at higher ionic strength. The tests can be applied to the study of a variety of pathologic states where immune complexes appear to play a role.     

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.     

The detection of circulating immune complexes containing immunoglobulin G

An immune complex assay using radiolabelled immunospecific antibodies against human IgG and polyethylene glycol precipitation (IgG-PEG assay) is described. The material reactive in this assay was evaluated using aggregated immunoglobulins, immune complexes prepared in vitro, sera of patients with a variety of disorders and normal human serum. Sucrose density gradient ultracentrifugation showed that only large-sized immune complexes (greater than 25 S) were detected. Comparison of the results of the IgG-PEG assay with those of the C1q binding assay showed a highly significant positive correlation (p less than 0.001). It was found that rheumatoid factors do not influence the results of the IgG-PEG assay. The method described in this study detects specifically immune complexes containing IgG and might be extended to the detection of other constituents of circulating immune complexes.     

Circulating immune complexes detected by 125I-Clq deviation test in sera of cancer patients.

The presence of circulating immune complexes in freshly drawn sera of patients with various forms of malignancies was detected by the 125I-Clq deviation test of Sobel et al. More than 50% of the 459 cancer sera showed a high inhibition of 125I-Clq uptake by sensitized sheep erythrocytes when compared with sera of 50 healthy laboratory personnel. The levels were compared with levels of total hemolytic complement and immunochemical determinations of Cl1 and C3. A correlation between high levels of circulating immune complexes and low levels of Clq was suggested. These immune complexes were separated by sucrose density gradient ultracentrifugation at low pH and were found to be heavier than 19S. Fluctuation of levels of immune complexes was evident when serial samples from the same patient were tested. Decrease of levels of immune complexes and a concomitant increase of Clq were detected after Calmette-Gueérin bacillus and autologous tumor cell treatment in some melanoma patients.     

Development of a sandwich ELISA to detect IgG and IgG sub-class antibodies specific for a major antigen (Ag 7) of Aspergillus fumigatus

A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub-classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin-prick test positive individuals. IgG sub-class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti-IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen-specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.     

The specific detection of IgG, IgA and the complement components C3 and C4 in circulating immune complexes

Four immune complex assays (PEG assays) are described; they are based on the binding of radio-labelled immunospecific antibodies to immune complexes containing IgG, IgA, C3 or C4, and subsequent precipitation with polyethylene glycol. Comparison of the results of the IgA-PEG assay with those of an existing immune complex assay (alpha-IgA-InhBA) showed that the former detects only large-sized (greater than 25 S) material. This was also demonstrated by sucrose density gradient ultracentrifugation of immune complexes in patients' sera as well as in preparations of aggregates containing IgA, C3 or C4. By using aggregates of mixed composition (IgG, IgA, C3 and C4) it was shown that each constituent could be detected by one of the 4 assays. Immune complexes containing the various constituents could also be detected in sera of patients with a variety of disorders. Further studies are needed to establish whether these constituents occur within the same complex or within different complexes.     

Immune response to B19 parvovirus and an antibody defect in persistent viral infection.

B19 parvovirus has been shown to persist in some immunocompromised patients, and treatment with specific antibodies can lead to decreased quantities of circulating virus and hematologic improvement. A defective immune response to B19 parvovirus in these patients was shown by comparison of results using a capture RIA and immunoblotting. In normal individuals, examination of paired sera showed that the dominant humoral immune response during early convalescence was to the virus major capsid protein (58 kD) and during late convalescence to the minor capsid species (83 kD). In patients with persistent parvovirus infection, variable titers against intact particles were detected by RIA, but the sera from these patients had minimal or no IgG to capsid proteins determined by Western analysis. Competition experiments suggested that this discrepancy was not explicable on the basis of immune complex formation alone and that these patients may have a qualitative abnormality in antibody binding to virus. In neutralization experiments, in which erythroid colony formation in vitro was used as an assay of parvovirus activity, sera from patients with poor reactivity on immunoblotting were also inadequate in inhibiting viral infectivity. A cellular response to purified B19 parvovirus could not be demonstrated using proliferation assays and PBMC from individuals with serologic evidence of exposure to virus. These results suggest that production of neutralizing antibody to capsid protein plays a major role in limiting parvovirus infection in man.