Autoantibodies in malaria, tuberculosis and hepatitis B in a west African population.

Following reports of associations between autoantibodies and living in the tropics, we have studied the seroprevalence and nature of anti-nuclear antibodies, anti-cardiolipin antibodies, antibodies to extractable nuclear antigens and anti-neutrophilic cytoplasmic antibodies in 351 West Africans with malaria, tuberculosis or hepatitis B, or in good health. Amongst healthy West Africans we found a seroprevalence of 7% for anti-nuclear antibodies with several staining patterns, and of 30.3% for anti-cardiolipin antibodies. Among patients with tuberculosis and malaria there was twice that frequency of anti-nuclear antibodies (predominantly speckled in pattern), and anti-neutrophil cytoplasmic antibodies (predominantly IgM) were demonstrated in a few cases. A possible association between IgG anti-cardiolipin antibodies and tuberculosis was observed (P < 0.05), but antibodies to double-stranded DNA were not elevated and no antibodies to extractable nuclear antigens were found in any of the patients or healthy individuals studied. Our findings suggest the need for caution in the interpretation of autoantibody tests in subjects from or living in the tropics, as well as in patients with tropical infections.     

Cutaneous and other lupus-like symptoms in carriers of X-linked chronic granulomatous disease: incidence and autoimmune serology

The objective of this study was to determine the utility of anti-nuclear antibody (ANA) testing in the investigation of cutaneous and other lupus symptoms in female carriers of X-linked chronic granulomatous disease (CGD). We undertook a prospective study of 19 carrier mothers attending our institution, with direct questioning of carriers concerning symptoms and testing for anti-nuclear and anti-phospholipid antibodies. A total of 58% reported significant photosensitive skin rashes, 42% reported mouth ulcers and 37% complained of joint pains that could not be attributed to other known causes. Anti-nuclear antibody (ANA) testing was negative in 73% of all carriers. The five positive ANAs were of low titre (maximum 1 : 320 on Hep 2 cells in two women) and only one weak positive double-stranded DNA antibody and no extractable nuclear antibodies were found. Several of the mothers, despite negative serology, benefited from referral to a specialist, and in some cases to specific treatment. A history of skin rashes, joint pain, fatigue and mouth ulcers should be sought actively in the female relatives of X-CGD patients but negative lupus serology should not preclude referral to appropriate dermatology or rheumatology services. as symptoms may respond well to appropriate treatment.     

An amplification system using BSA-antibody conjugate for sensitive enzyme immunoassay

A procedure is described for sensitive titration of antibodies, macromolecular antigens and haptens by enzyme immunoassay. It involves using first antigen or antibody labelled with bovine serum albumin (BSA) and then an anti-BSA antibody conjugated with an enzyme. The performance characteristics of this assay are indicated and compared with those for conventional enzyme immunoassay. The present procedure allowed fast sensitive titration of human IgE, rabbit type III anti-streptococcal antibody and cAMP.     

Development of the antinuclear and anticytoplasmic antibody consensus panel by the Association of Medical Laboratory Immunologists

The Association of Medical Laboratory Immunologists (AMLI) have developed a panel of antinuclear and anticytoplasmic antibody consensus sera that can be useful for enzyme immunoassay (EIA), Ouchterlony, and immunofluorescence assay methods. It was developed to assist in the evaluation of newly available EIA methods for the detection of autoantibodies. The panel of sera was evaluated in several clinical laboratories and a large number of laboratories owned by manufacturers of clinical autoantibody testing kits. The majority of sera performed well for the EIAs in both the clinical laboratories and the manufacturers' laboratories, but some samples had discrepant results. A major source of discrepancy is the current inability of the EIA results to be directly compared in a quantitative way as no standardization exists. The evaluation demonstrated lower sensitivity of detection by the Ouchterlony method. The limited evaluation of the sera with immunoblotting and Western blotting did not show good agreement with other methods. Further work must be done to standardize blotting methods prior to their use in routine clinical testing. The sera are now available to vendors and clinical laboratories for use in the detection of SS-A, SS-B, Sm, U1-RNP, Scl-70, Jo-1, double-stranded DNA, and centromere antibodies. The availability of the consensus sera will help evaluate and improve the EIA methods currently being used.     

Development of the Antinuclear and Anticytoplasmic Antibody Consensus Panel by the Association of Medical Laboratory Immunologists

The Association of Medical Laboratory Immunologists (AMLI) have developed a panel of antinuclear and anticytoplasmic antibody consensus sera that can be useful for enzyme immunoassay (EIA), Ouchterlony, and immunofluorescence assay methods. It was developed to assist in the evaluation of newly available EIA methods for the detection of autoantibodies. The panel of sera was evaluated in several clinical laboratories and a large number of laboratories owned by manufacturers of clinical autoantibody testing kits. The majority of sera performed well for the EIAs in both the clinical laboratories and the manufacturers' laboratories, but some samples had discrepant results. A major source of discrepancy is the current inability of the EIA results to be directly compared in a quantitative way as no standardization exists. The evaluation demonstrated lower sensitivity of detection by the Ouchterlony method. The limited evaluation of the sera with immunoblotting and Western blotting did not show good agreement with other methods. Further work must be done to standardize blotting methods prior to their use in routine clinical testing. The sera are now available to vendors and clinical laboratories for use in the detection of SS-A, SS-B, Sm, U1-RNP, Scl-70, Jo-1, double-stranded DNA, and centromere antibodies. The availability of the consensus sera will help evaluate and improve the EIA methods currently being used.     

Immunoassay for serologic diagnosis of influenza type A using recombinant DNA produced nucleoprotein antigen and monoclonal antibody to human IgG

Influenza type A nucleoprotein (NP) derived from the full length cloned gene expressed in E. coli was evaluated in a solid phase enzyme immunoassay (EIA) for detection of human antibody to influenza. Monoclonal antibody to human IgG was used for detection. Direct and indirect assays were developed and sera were tested in serial and single dilution formats. Preliminary results indicated that recombinant-and virion-derived NP antigens were comparable in binding ability to plastic and binding human antibody. Eighty-seven paired sera from influenza patients were tested. The most sensitive assay (indirect-serial dilution) detected 56 (64%) rises and the simplest assay (direct-single dilution) detected 43 (49%) rises, compared to 36 (41%) for complement fixation. Paired sera from 18 control patients showed no evidence of antibody rises by any of the assays. Forty-nine paired sera from influenza B infected patients were negative for antibody rises except for one borderline rise by the indirect-serial dilution assay. These results indicate that the use of recombinant DNA derived nucleoprotein for immunoassay is feasible. The sensitivity of immunoassays using NP adsorbed to the solid phase and monoclonal antibody specific for human IgG to detect bound antibody exceeded that of conventional complement fixation testing for establishing serologic evidence of influenza type A infection.     

Comparison of methods for detection of serum antibody to murine rotavirus.

Mice are frequently used as animal models for the study of rotaviral infections. Since natural infection is common in laboratory mice, it is important that rotaviral studies, as well as other studies utilizing suckling mice, employ animals of known immune status to murine rotavirus. A variety of homologous and heterologous enzyme immunoassay systems and an immunofluorescence technique were thus compared to determine the immunoassay that is most effective at detecting adult mice seropositive for rotaviral antibody. It was determined that a homologous enzyme immunoassay inhibition technique utilizing murine rotavirus-derived reagents was the most efficient serologic assay evaluated. A serologic response was consistently detected by this assay by 5 days after experimental rotaviral inoculation of adult mice. A homologous antibody-binding enzyme immunoassay, a heterologous inhibition enzyme immunoassay utilizing antigenically related simian rotavirus (SA-11) reagents, and an immunofluorescence technique utilizing Nebraska calf diarrhea virus antigens were found to be less sensitive for detecting serum antibody to murine rotavirus.     

Antibody response to cytomegalovirus polypeptides captured by monoclonal antibodies on the solid phase in enzyme immunoassays.

Antibodies to different cytomegalovirus (CMV) polypeptide antigens, captured by monoclonal antibodies coated on the solid phase of an enzyme immunoassay test, were analyzed in 42 serum pairs submitted for serodiagnosis of CMV infection. Three CMV antigens, captured on the solid phase by three monoclonal antibodies of different specificities, designated CH92-1, CH65-1, and CH16-1, were glycoproteins A (gA), gC, and gD, respectively; and one antigen, captured by CH23, was a polypeptide with an apparent molecular weight of 150,000, possibly associated with the nucleocapsid. Of these four CMV antigens, gA captured by CH92-1 was most effective in eliciting an antibody response. Antibody to this antigen was present in serum samples at a higher concentration in primary and reactivated infection and persisted longer than did antibody to the other tested antigens. In contrast, antibody to antigen captured by CH23 was at a lower concentration, rose more slowly in infection, and persisted for a shorter time than did antibody to the other antigens. Antibody response to gC and gD was intermediate in concentration and temporal appearance compared with the antibody response to gA and to the polypeptide bound by CH23. An enzyme immunoassay on paired serum samples with the captured glycoproteins as antigen was equal for the detection of current infection to an enzyme immunoassay with the whole CMV antigen from infected cell lysates. Enzyme immunoassays with either the CMV glycoproteins or the whole CMV antigen from infected cell lysates were superior to a complement fixation test with a glycine extract antigen for serodiagnosis of current infection.     

DEVELOPMENT OF AN EFFICIENT AND SIMPLE METHOD FOR CONJUGATION OF LACCASE TO IMMUNOGLOBULIN AND ITS CHARACTERIZATION BY ENZYME IMMUNOASSAY

A new laccase-conjugated antibody is developed, containing laccase as an enzyme marker, obtained from culture supernatant of the basidiomycetous fungi Pleurotus ostreatus. The efficacy of the laccase-conjugated antibody was demonstrated in indirect and direct enzyme immunoassay after using periodate and glutaraldehyde conjugation methods. The assay based on laccase-conjugated antibody is potent to detect antigens when compared with peroxidase conjugated antibody.     

Histone and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence.

A method of swelling spermatozoa and other cells, which leads to the exposure of nuclear antigens is described. By applying the indirect IFT on these swollen cells with sera containing antibodies to nuclear antigens, and by comparing the results to those obtained in other tests (measuring anti-nuclear antibodies), the following conclusions could be drawn: (a) By swelling human spermatoza, nuclear antigens of the sperm are exposed, and can be used for the detection of antibodies directed against them. (b) Heterlogous antibodies to histones F2al, FIa2 and F3 which can not be detected in the indirect IFT on rat liver cells, become detectable after swelling of these cells. (c) Mature human spermatozoa contain, in addition to double-stranded DNA and protamine, small amounts of histone F2b and F2a2. (d) In mature human spermatozoa histone F1 is absent.     

Enzyme immunofiltration technique for rapid diagnosis of herpes simplex virus eye infections in a rabbit model.

A rapid enzyme immunofiltration assay for herpes simplex virus (HSV) has been developed which is sensitive enough to detect viral antigens in eye swabs from rabbits with primary herpes keratitis. This assay employs a specially designed filter manifold to immobilize whole cells and cell debris dissociated from the swabs. Viral antigens trapped on the filters are then detected in an indirect immunoassay utilizing staphylococcal protein A conjugated with horseradish peroxidase. The assay required only 2.5 h to perform and could be read visually. Reconstruction experiments indicated that antigen from as few as 49 HSV-infected cells could be detected. Calcium alginate swabs were shown to recover more viral antigen than dacron swabs. The enzyme immunofiltration assay detected HSV antigens on 95% of the eye swabs from which infectious virus was recovered. In addition, HSV antigen was also detected in several swabs from infected eyes which did not yield infectious virus, presumably because the virus was neutralized by native antibody present in the lacrimal fluid. This enzyme immunofiltration assay technique lends itself to the elution of native antibody bound to the viral antigens, and this may be especially applicable in the diagnosis of recurrent HSV keratitis, where antiviral antibody in the lacrimal fluid may interfere with virus isolation and fluorescent-antibody or other virus detection assays.     

Immunochemical studies of a murine polyreactive IgG2b autoantibody with rheumatoid factor activity

The hybridoma, 62H3, which secretes a monoclonal IgG2b with anti-HLA-DR specificity, was expanded in pristane-primed BALB/c mice and the antibody was isolated from the ascitic fluid by affinity chromatography on Protein A-Sepharose. The purified IgG2b antibody was tested by an enzyme immunoassay for antibody activity against a panel of 40 self and non-self antigens. It was found to react strongly with beta-galactosidase, actin, glutamate dehydrogenase, rabbit and human IgG and di- and trinitrophenyl groups; and moderately with tubulin, insulin and phosphorylcholine; but it did not react with various other self and non-self antigens, such as DNA, albumin, keyhole limpet hemocyanin, hen lysozyme and horseradish peroxidase. Fab and Fc fragments were prepared from this IgG2b by papain proteolysis. The Fab fragment possessed the same spectrum of polyreactivities as the native IgG2b, whereas no activity was detected with the Fc fraction. In order to investigate the properties of the antigen binding site, the actin, TNP and rabbit IgG antibody activities were studied in more detail by enzyme immunoassay, Western blot and immunocytochemistry. The monomolecular nature of this multireactivity was confirmed by immunoabsorption analysis. Furthermore, 62H3 monoclonality was also verified by comparative isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with other monospecific antibodies. The dissociation constants (Kd) of antigen-antibody equilibria in solution were measured. The Kd for actin was 1.11 +/- 0.24 x 10(-5) M and the Kd for TNP-BSA was 8.7 +/- 0.51 x 10(-7) M. No interaction with rabbit IgG could be detected in solution. These findings raise the question of the possible implication in autoimmune pathology or in normal physiology of IgG class polyspecific antibodies with solid-phase restricted cross-reactive rheumatoid factor activity.     

Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes.

The Raji cell assay is regarded as a test for the detection and quantitation of immune complexes. It is frequently positive in sera from patients with SLE. We have demonstrated a relationship between Raji cell binding and antibodies to DNA and soluble cellular antigens. In five sera containing high titres of antibodies of known single specificity, most of the Raji cell binding occurred in the 7S IgG fraction where the majority of anti-nuclear antibody was also found. When each of these sera was incubated with its specific antigen, Raji cell binding increased. Subsequent fractionation showed that this binding was in the high molecular weight fraction (greater than 200,000 daltons) and that Raji cell binding and antibody activity were abolished in the 7S fraction. These data confirm that Raji cell bind immune complexes but also indicate that 7S anti-nuclear antibodies may interact directly with Raji cells by an unknown mechanism. Therefore, in sera of patients with anti-nuclear antibodies, binding to Raji cells does not necessarily imply the presence of immune complexes alone.     

Detection of human immunodeficiency virus type 1 antibody by using commercially available whole-cell viral lysate, synthetic peptide, and recombinant protein enzyme immunoassay systems.

A total of 575 serum specimens received for human immunodeficiency virus type 1 (HIV-1) antibody determination were tested prospectively by enzyme immunoassay with a whole-cell viral lysate (VL) (Genetic Systems Corp.), a synthetic peptide (SP) (United Biomedical, Inc.), and a recombinant protein (RCP) (Syva Co.). Concordance of all three antigens was noted for 559 of 575 (97.2%) specimens tested. Of the specimens tested, 90 (15.7%) were positive and 469 (84.3%) were negative. Retrospective testing by SP and RCP of 86 specimens indeterminate for HIV-1 antibody by VL, Western blot (immunoblot), and immunofluorescence was also performed. The results of both phases of this study indicate that the specificity of the three antigens is RCP greater than SP greater than VL. As noted from the prospective phase, the sensitivities of the antigens appear to be equivalent, indicating that the RCP and SP systems could be used in place of, or to confirm, a VL enzyme immunoassay screening test.     

Computer-assisted pattern recognition of autoantibody results

Immunoassay-based anti-nuclear antibody (ANA) screens are increasingly used in the initial evaluation of autoimmune disorders, but these tests offer no "pattern information" comparable to the information from indirect fluorescence assay-based screens. Thus, there is no indication of "next steps" when a positive result is obtained. To improve the utility of immunoassay-based ANA screening, we evaluated a new method that combines a multiplex immunoassay with a k nearest neighbor (kNN) algorithm for computer-assisted pattern recognition. We assembled a training set, consisting of 1,152 sera from patients with various rheumatic diseases and non-diseased patients. The clinical sensitivity and specificity of the multiplex method and algorithm were evaluated with a test set that consisted of 173 sera collected at a rheumatology clinic from patients diagnosed by using standard criteria, as well as 152 age- and sex-matched sera from presumably healthy individuals (sera collected at a blood bank). The test set was also evaluated with a HEp-2 cell-based enzyme-linked immunosorbent assay (ELISA). Both the ELISA and multiplex immunoassay results were positive for 94% of the systemic lupus erythematosus (SLE) patients. The kNN algorithm correctly proposed an SLE pattern for 84% of the antibody-positive SLE patients. For patients with no connective tissue disease, the multiplex method found fewer positive results than the ELISA screen, and no disease was proposed by the kNN algorithm for most of these patients. In conclusion, the automated algorithm could identify SLE patterns and may be useful in the identification of patients who would benefit from early referral to a specialist, as well as patients who do not require further evaluation.     

Microbes and Autoimmunity

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.     

Nephritogenic antibodies bind in glomeruli through interaction with exposed chromatin fragments and not with renal cross-reactive antigens

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.     

Specificity of anti-nuclear antibodies induced in F1 mice undergoing the graft-vs-host reaction: isotypes and cross-reactivities.

(C57BL/6 X DBA/2)F1 mice undergoing the graft-vs-host reaction (GVHR) produce autoantibodies after the injection of DBA/2 lymphoid cells. The anti-nuclear antibodies, including anti-poly (ADP-ribose) and anti-extractable nuclear antigens (ENA), in the sera of the autoimmune GVH F1 mice were investigated. Antibodies to double-stranded DNA, single-stranded DNA and ENA were predominantly IgG. In contrast, the autoantibodies to poly(ADP-ribose) were both IgG and IgM, although the former was predominant. These autoantibodies induced by the GVHR showed similar cross-reactivities with a number of nucleic acids to the monoclonal and some serum antinuclear antibodies derived from mice or humans with systemic lupus erythematosus (SLE). These results support the idea that GVH F1 mice are a good model of human SLE.     

Enzyme immunoassay inhibition assay for the detection of rat rotavirus-like agent in intestinal and fecal specimens obtained from diarrheic rats and humans.

An enzyme immunoassay inhibition assay was developed to detect rat rotavirus-like agent (RVLA) or antigenically related viruses in intestinal washings and homogenates obtained from diarrheic rats and in fecal specimens obtained from diarrheic humans. In this assay, RVLA antigens in a test sample inhibited the binding of a biotin-labeled anti-rat RVLA antibody preparation to rat RVLA antigens bound to the solid phase. Intestinal washings and homogenates obtained 1 day after RVLA infection of suckling rats inhibited the binding of the biotin-labeled antibody to the solid-phase rat RVLA antigens by 76 to 100%. The inhibition was blocked by RVLA immune rat serum but not by nonimmune rat serum. Of 27 fecal specimens obtained from diarrheic humans, 6 produced disease characteristic of rat RVLA infection when inoculated into suckling rats. Four of these six specimens produced greater than 50% inhibition in the enzyme immunoassay. Fecal specimens obtained from diarrheic humans that were determined to be negative for RVLA produced an average inhibition of 9.2%. This enzyme immunoassay appears to be a useful diagnostic and research tool for the study of infections with at least one of the antigenically distinct rotaviruses.     

Cross-reactivity of mouse monoclonal antibodies produced by in vitro or in vivo immunization

The effect of different immunization schemes on the resulting antibody specificity was investigated. The cross-reactivity of monoclonal antibodies produced by in vitro vs. in vivo immunization was tested, using a solid phase enzyme immunoassay. Ten different monoclonal antibodies were tested against 15 different antigens. There was no difference in cross-reactivity between the two types of antibodies when tested against antigens coated onto the plastic wells in a buffer solution.When the antigens were dried onto the plastic wells the IgM monoclonal antibodies produced by in vitro immunization exhibited a somewhat different reactivity pattern. However, the assay design was shown to be of more importance than the immunization procedure when determining antibody specificities.