Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.     

Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis.

MPB70 is a highly species specific protein which is secreted from Mycobacterium bovis during culture. To investigate whether antibodies against MPB70 can be used as an indicator of infection with M. bovis, an enzyme-linked immunosorbent assay was developed, based on the use of biotinylated protein G, to provide a common indicator for antibody formation in different species. During experimental infection with M. bovis in cattle, a characteristic pattern of anti-MPB70 antibody production was observed with an initial flat plateau followed by a marked rise 18 to 20 weeks after infection. Skin testing with bovine tuberculin purified protein derivative (PPD), which was shown to contain antibody-reactive MPB70, was a potent stimulator of antibody production in infected animals. In experimentally infected cattle, we observed an inverse relationship between antibody activity and delayed-type hypersensitivity skin test reactions. In natural M. bovis infections, skin testing with PPD was also a potent stimulator of anti-MPB70 formation. Comparison between the enzyme-linked immunosorbent assay for antibodies to MPB70 and that for antibodies to the widely cross-reacting M. bovis BCG antigen 85B in animals with M. bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Corynebacterium pseudotuberculosis infections showed that formation of antibody to MPB70 was highly specific for infection with M. bovis. The use of an MPB70-containing PPD preparation for skin testing followed by this anti-MPB70 assay is a highly specific indicator of M. bovis infection. Adjustment of the test conditions is expected to provide an increased sensitivity of the procedure for the diagnosis of natural M. bovis infections.     

Cloning of a species-specific antigen of Mycobacterium bovis.

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.     

Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ～90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.     

Biological Activity in Sensitized Guinea Pigs of MPB 70, a Protein Specific for Some Strains of Mycobacterium bovis BCG

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.     

Early antibody responses to experimental Mycobacterium bovis infection of cattle

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.     

Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG.

The immunogenic proteins MPB64 and MPB80 of Mycobacterium bovis BCG were purified to homogeneity and compared with MPB70. MPB70 and MPB80 showed a similar distribution in substrains of BCG, both being present in high concentrations in culture fluids of BCG substrain Tokyo, BCG Moreau, BCG Russia, and BCG Sweden and in only very small amounts in BCG Glaxo, BCG Tice, BCG Copenhagen, and BCG Pasteur. In various physicochemical properties MPB70 and MPB80 were closely similar, but MPB80 had a distinctly lower pI value. The N-terminal amino acid sequence was identical for the first 30 residues. In reactions with anti-MPB70 antibodies and delayed-type hypersensitivity skin reactions, MPB70 and MPB80 also had very similar properties. These results show that MPB70 and MPB80 are two closely similar forms of the same gene product, and postsynthetic changes probably explain the observed differences. By contrast, MPB64 had a higher molecular weight. The N-terminal amino acid sequence showed no homology with MPB70, and these two proteins showed no immunologic similarity. MPB64 and MPB70 showed only very restricted cross-reactivity with other species of mycobacteria but cross-reacted with Nocardia asteroides. The similar occurrence in eight different substrains of BCG indicated that the two proteins are influenced by similar control mechanisms, but in contrast to MPB70, MPB64 occurred in sufficient concentration in two strains of Mycobacterium tuberculosis to give a distinct spot in two-dimensional polyacrylamide gel electrophoresis of their culture fluids.     

Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle

The gene encoding Babesia bovis rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant B. bovis RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between B. bovis-infected sera and B. bigemina-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to B. bovis.     

Immunochemical Characterization of the MPB70/80 and MPB83 Proteins of Mycobacterium bovis

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.     

Systemic and cutaneous mucus antibody responses of channel catfish immunized against the protozoan parasite Ichthyophthirius multifiliis

Fish acquire protective immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection or inoculation with I. multifiliis immobilization antigens (i-antigens). In both cases, parasite-immobilizing antibodies have been identified in sera and mucosal secretions. To investigate the kinetics of this immune response, antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in the sera and cutaneous mucus of channel catfish (Ictalurus punctatus) that were either infected with parasites or given a single injection of purified i-antigen (5.0 microg/fish) in Freund's incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A405) value of 0.54 +/- 0.17 and 0.35 +/- 0.03, respectively, which were significantly higher (alpha = 0.05) than the pretreatment serum (1:80 dilution) antibody absorbance value of 0.24 +/- 0.05. At 14 weeks, mean serum (1:80 dilution) ELISA absorbance values in the teo groups of fish increased to 0.79 +/- 0.30 and 0.71 +/- 0.24, respectively. In both groups of fish, antibody levels in cutaneous mucus (undiluted) were much lower than those in sera. Infected fish had detectable mucus (undiluted) antibody levels from 3 to 9 weeks, with the highest mean value (0.30 +/- 0.07) occurring at 7 weeks. Although individual inoculated fish produced serum antibody absorbance values comparable to those seen in infected fish, the mean mucus antibody values in this group did not rise above pretreatment levels. I. multifiliis infection induced a transient mucosal antibody response that coincided with the resolution of infection. Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously.     

Fluorescence Polarization Assay and SDS-PAGE Confirms Matrilysin Degrades Fibronectin and Collagen IV whereas Gelatinase: A Degrades Collagen IV but not Fibronectin

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.     

Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.     

Improvement for identification of Mycobacterium tuberculosis complex by immunochromatographic assay anti-MPB 64 monoclonal antibodies with "H2O2" addition trial on blood culture samples

An improvement trial for identification of the Mycobacterium tuberculosis complex by immunochromatographic assay using anti-MPB 64 monoclonal antibodies (MPB 64-ICA) is documented. The new MPB 64-ICA is very useful for simple and rapid identification of the Mycobacterium tuberculosis complex with specificity, but it is a little difficult to prove the Mycobacterium tuberculosis complex on the blood culture samples taken from the immunosuppressive or the AIDS patients, because the reactive bands are red or reddish-purple in both the control and positive zones of immunochromatographic assay, which are often indistinguishable from the background mixed with hemoglobin caused from the blood samples. We tried to remove the hemoglobin interference in the samples and was able to make clear the background with addition of hydrogen peroxide (H2O2). The results are followings; the optimum time and density of H2O2 are from 5 to 15 minutes and 35%, respectively. Accordingly, the improvement trial for identification of the Mycobacterium tuberculosis complex by immunochromatographic assay using MPB64-ICA with H2O2 is useful to identify the positive reactive band from the interference by hemoglobin.     

Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.     

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibodies to porcine parvovirus (PPV). Antisera to PPV were raised in pigs, for which PPV grown on PK15 cells was used for primary intranasal inoculations, and PPV cultured on autologous kidney cells for booster immunisations. A competition ELISA was developed, based on the principle of a double antibody sandwich assay, using immunoglobulin fractions prepared from these sera. The ELISA was compared with a haemagglutination inhibition (HI) test. The tests were equally sensitive for detecting antibodies early after infection and for detecting a significant increase in antibody titre between paired sera. A high correlation was found between antibody titres of field sera measured by the two tests (r = 0.91). We conclude that ELISA is preferable to the HI test, because it is labour-saving and can be standardised better and automated.     

Enzyme-linked immunosorbent assay of bovine tuberculosis by crude mycobacterial protein 70

MPB70 (mycobacterial protein of BCG 70) as T-cell stimulator has been tried with an intradermal skin test (IST) and enzyme-linked immunosorbent assay (ELISA) of bovine tuberculosis (BTB). In this study, crude mycobacterial protein 70 (CMP70) was prepared by anion exchange chromatography from the culture supernatant of Mycobacterium bovis AN5 and CMP70 ELISA was compared with purified protein derivative (PPD) ELISA. PPD and CMP70 ELISA have shown a positive reaction to the sera of M. bovis infected cattle and IST positive reactors. One of three IST negative cattle showed the nonspecific reaction in PPD ELISA, whereas all of the IST negative cattle (n=3) were did not show the nonspecific reaction in CMP70 ELISA. When each ELISA was applied to sixty-two IST positive cattle, ELISA positive reactors were eighty four per cent to CMP70 antigen and fifty-two per cent to PPD. CMP70 has been shown to be more specific and sensitive than PPD in ELISA.     

Purification of a Major Mycobacterium bovis Antigen for the Diagnosis of Bovine Tuberculosis

A Mycobacterium bovis antigen has been purified from culture filtrate by chromatofocusing. This antigen is a major component of culture filtrate and cell extracts and shows a considerable degree of micro-heterogeneity in electric charge and molecular weight. Studies with monoclonal and polyclonal antibodies raised against the purified antigen show that some of its antigenic determinants also occur in higher molecular weight species in culture filtrate and particularly in whole cell preparations. Immunoblotting and ELISA studies, using sera from M.bovis-infected animals, showed that this antigen is one of the most immunoreactive components of M. bovis, recognized by the majority of animals with detectable antibody response to M. bovis. The specificity of the purified antigen is far superior to that of the crude culture filtrate, with very few false positive results. The purified antigen also elicits strong in vivo and in vitro cell-mediated responses. The amino acid compositions of two variants of this antigen have been determined and found to be similar to that of MPB-70.     

Enzyme-linked immunosorbent assay for titration of Haemophilus influenzae capsular and O antigen antibodies.

The enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of immunoglobulin M (IgM) and IgG antibodies against capsular and O antigens of Haemophilus influenzae. Purified capsular polysaccharide and lipopolysaccharide were used as antigens, with optimal coating concentrations being about 50 and 100 micrograms/ml, respectively. The antibody content was expressed as the highest serum dilution (-log10) showing an absorbance of 0.2 above the background level. The titers of hyperimmune sera (reference sera) ranged between 5 and 7 -log10. The sensitivity of the method was about 80 ng/ml with regard to anticapsular antibodies and 3 to 5 ng/ml with regard to anti-lipopolysaccharide antibodies. For detection of antibodies against capsular polysaccharide in sera obtained after primary immunization, ELISA was about 100-fold more sensitive than the indirect hemagglutination assay, whereas in hyperimmune sera, ELISA was about 10-fold more sensitive than the indirect hemagglutination assay. The sensitivity of ELISA for detecting anticapsular antibodies after primary and booster immunizations was 50-fold higher than that of the bactericidal assay using capsulated bacteria, whereas the sensitivity of the two methods was the same when hyperimmune sera were tested. ELISA performed with lipopolysaccharide as the antigen was about 50- and 150-fold more sensitive than the complement fixation and bactericidal assays tested with noncapsulated variants after primary injection and hyperimmunization, respectively.     

Specificities and characteristics of mitochondrial protein antigens assessed by enzyme linked immunosorbent assay for anti-mitochondrial antibodies. Relationship to immunofluorescent and precipitating antibodies.

M-A, M-B and M-C are autoantibodies to mitochondrial proteins frequently found in primary biliary cirrhosis. To study the characteristics and specificities and to isolate antigens reacting with the autoantibodies a sensitive assay (ELISA) was established. Using this technique a significantly elevated level of antibodies was detected in patients with primary biliary cirrhosis (mean OD 460.2, s.d. 145.8) compared to normals (133.7 +/- 49.7). The assay correlated well with the indirect immunofluorescence test for detecting anti-mitochondrial antibodies and the mitochondrial fluorescence could be abolished by absorption of autoantibodies with the mitochondrial fraction. All three types of antibodies could be detected by ELISA; however, sera containing the combination of two or more antibodies yielded higher ELISA values. The ELISA confirmed that the M-A antigen is trypsin and acid (pH 3.0) sensitive but DNAase and RNAase resistant while the M-B antigen is DNAase and trypsin sensitive. The antigens were enriched in the supernatant isolated from the mitochondrial fraction centrifuged at 1,800 g for 60 min and 2% polyethylene glycol precipitates of the mitochondrial fraction. The antigens were found phosphate buffer soluble and therefore could also be enriched by phosphate buffer extraction of the mitochondrial proteins. Thus, ELISA described here provided a sensitive method in the assessment of characteristics and purification of autoantigens related to mitochondrial antibodies.     

Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

A new solid-phase fluorescence immunoassay technique is described and is exemplified by the detection of murine monoclonal antibodies to human IgG in hybridoma culture supernatants and the detection of murine IgG. The assay is performed in a specially designed 96-well plate. For antibody detection, antigen bound to submicron polystyrene particles is bound to its specific antibody, which is in turn reacted with fluorescein-labeled affinity-purified goat anti-mouse IgG. The reaction is complete in 10 min at ambient temperature. The solid phase is separated from the reaction mixture by filtration, washed and the total particle-bound fluorescence is determined by front-surface fluorimetry. The sensitivity of the technique for antibody detection is equivalent to enzyme-linked immunoabsorbent assay and 2-4 ng/ml for murine IgG detection. It is readily amenable to automation.