Use of antisperm antibodies in differential display Western blotting to identify sperm proteins important in fertility

BACKGROUND: Antisperm antibodies (ASA) may be an important cause of infertility but current tests for the detection of ASA have poor prognostic value. The inadequacy of current tests may reflect the inability of these tests to define the antigenic specificity of the sperm proteins with which the ASA react. Identification of the sperm proteins that ASA bind to is a necessary preliminary step to the development of more useful diagnostic tests for ASA. METHODS: A sensitive Western blotting technique was used to compare the antigenic specificities of ASA from men who were infertile (n = 6) with those who were fertile following vasectomy reversal (n = 3). Normal fertile men (n = 3) and infertile men with known ASA (n = 4) were also included in the analysis. RESULTS: All men, including the normal fertile controls, had ASA detectable in our system. Several sperm proteins were identified that react with ASA from infertile but not fertile men. Quantitative differences in the binding of ASA to some proteins were also demonstrated. Additionally, we demonstrated that normal motile sperm are coated with an antibody that appears to be bound to sperm by a non-antigenic mechanism. CONCLUSION: Sera from all men contained ASA, but clearly some of these did not cause infertility. Characterization of the proteins that are antigens for ASA from infertile but not fertile men may allow the development of more accurate tests for infertility-inducing ASA. The significance of immunoglobulin G coated on normal sperm remains to be determined.     

Liposomal immunoassay as a method for detecting microorganism antigens and their antibodies]

The paper presents experimental data on the use of the liposomal immunoassay (LIA) with a fluorescence marker to detect lipopolysaccharide antigens (LPS-AG) of the causative agents of infectious diseases (S. typhimurium, S. typhi, F. tularensis) and antibodies to them in the model systems and human serum. The sensitivity of determination of specific antibodies to LPS-AG is shown to be 15-160 times as high as that of RPGA and the sensitivity of determination of LPS-AG is comparable to that of solid-phase enzyme immunoassay. The stability and storage of diagnostic immunoliposomal test systems are dealt with. It is shown that the liposomal diagnostic agents can be stable without losing their properties for years. Whether LIA is of diagnostic value in detecting salmonellosis in children in the clinical setting is discussed and the value of this assay is compared with that of other laboratory methods. The data on how LIA can be automated are presented. Its analytical advantages in using in laboratory diagnosis are discussed.     

Micro mixed hemadsorption assay--a sensitive method for detecting antibodies against tumor-associated antigens.

Antibodies against methylcholanthrene (MCA)-induced sarcomas in Fischer (F344) rats were detected by a modified micro mixed hemadsorption (MHA)-assay. The assays detected anti-tumor antibodies as titers up to 1 : 320 in sera from hyperimmunized rats and at titers up to 1 : 160 in sera from rats bearing a 5-8 cm3 progressively growing tumor. MHA-titers decreased when sera were absorbed with sarcoma cells prior to MHA-assays. IgG antibodies in sera from tumor bearing rats showed titers of 1:20 and 1:5. These anti-tumor sera formed rosettes on the corresponding sarcoma cells as well as other sarcomes induced by MCA in F344 and Lewis strain rats tested. The assay was modified for a micro technique using a microtest plate (No. 3034, Falcon, CA). This modification yielded as assay requiring only 10 microliter of test sera. The test is quantitative and highly sensitive and results are reproducible. Several critical factors which influence test results in this assay were examined.     

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-l-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4°C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.     

Enzyme-linked immunosorbent assay (ELISA) for detecting infectious laryngotracheitis viral antibodies in chicken serum

The conditions of an indirect-enzyme linked immunosorbent assay for infectious laryngotracheitis virus (ILT) antibodies have been established. The specificity of the reaction was demonstrated. The method offers a simple and specific antibody assay for the detection of antibodies to ILT virus arising from vaccination or challenge infection.     

Western-blotting method for detecting antibodies against human muscle contractile proteins in myositis

Many investigators report anti-muscle antibodies using various kinds of methods. The Western-blotting method, however, has not previously been used for this purpose. We have detected antibodies to muscle contractile proteins in sera from patients with collagen disease and muscular dystrophy by this method. The antigens detected included myosin heavy and light chains, tropomyosin and troponin complex. Our method is a quick and sensitive way to determine which are the antigenic muscle contractile proteins.     

Avidin-biotin latex agglutination assay for detection of antibodies to viral antigens.

A new method for attaching antigens to latex by an avidin-biotin technique is described. The procedure permits control of the amount of antigen attached to the latex and eliminates the need for highly purified antigens and destructive bridging chemicals. The avidin-biotin latex agglutination assay is a simple, rapid test well suited to detection of viral antibody. The sensitivity and specificity, respectively, of the avidin-biotin latex agglutination assay compared with other assay results for antibody to viruses were as follows: cytomegalovirus, 98 and 100% (indirect hemagglutination assay); measles virus, 96 and 100% (enzyme-linked immunosorbent assay); and herpes simplex virus, 78 and 100% (indirect hemagglutination assay).     

A simple method for detecting antibodies to rubella.

A simple microplate method of enzyme linked immunosorbent assay for Rubella antibody is described. This Micro-ELISA was compared with haemagglutination inhibition in a study of 188 human sera. The total discrepancy rate between the two tests was only 3-7%.     

Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens

Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody.     

Use of latex agglutination technique for detecting Legionella pneumophila (serogroup 1) antibodies.

Following the outbreak of Legionnaires' disease in Stafford in 1985, 500 serum samples were submitted to the indirect immunofluorescence antibody test and a latex agglutination. Latex agglutination using ultrasonically disrupted Legionella pneumophila antigens coupled to latex particles, proved a rapid, simple method for detecting circulating antibodies to L pneumophila in a one minute slide latex agglutination test. There was good correlation with the indirect immunofluorescence antibody test (IFAT), and the specificity and sensitivity with respect to a diagnostic result were 98.3% and 97.6%, respectively, using a series of well characterised sera. The latex agglutination test seems well suited as a screening test for presumptive cases of Legionnaires' disease; the latex reagent is easy to prepare and seems to remain stable at 4 degrees C for up to six months.     

Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

Genetic immunization maps T cell (auto)immune responses to self antigens homologous to exogenous proteins

Genetic immunization represents a new tool for investigating physiological and pathological immune responses. Here we used genetic immunization with naked DNA to study the immune relevance of aminoacid sequence homologies by evaluating the outcome of immunization to a viral protein homologous to an HLA molecule. The viral protein was Balf2, a protein of Epstein-Barr virus (EBV) that shares aminoacid sequence homology with the HLA allele DRB1*0801. After genetic immunization of BALB/c mice with a construct encoding Balf2, we analyzed T cell responses of immunized mice. We found that cross-reactive proliferative and cytotoxic responses were raised to the homologous sequence as expressed by HLA-DRB1*0801. Furthermore, preferential secretion of Th1-type pro-inflammatory cytokines occurred. This strategy can allow rapid screening of interactive immune networks involving aminoacid sequence homologies between organisms.     

An immunoenzymatic staining assay (ISA) for the rapid screening of monoclonal antibodies detecting membrane and cytoplasmic antigens

We report a rapid and very sensitive immunoenzymatic staining assay (ISA) for the determination of the fine specificity of monoclonal antibodies directed against cellular antigens. Reactivity is analysed at the single cell level by light microscopy using alkaline phosphatase-conjugated second and third antibodies on cells bound to Terasaki plates. Reactive cells can be defined by their morphology even in preliminary screening procedures. Only small numbers of cells are necessary for this assay which is comparable to radioimmunoassays in its sensitivity. Plates with fixed cells can be stored at 4 degrees C so that a permanent 'library' of various cell types is always immediately available for use. The test is also suitable for human blood cell phenotyping. Moreover, a simple modification of the procedure by short pretreatment of the cells with the detergent Brij allows the detection of antigens within the cytoplasm. The importance of evaluating the cytoplasmic compartment in order to define antibody specificity and study the distribution of different cytoplasmic and membrane antigens is emphasized.     

The use of colloidal gold-labelled antibodies for demonstrating viral antigens

The principal possibility of using antibodies conjugated with colloid gold for better visualization of viruses, viral antigens, and their immune complexes in electron microscope was studied on the experimental model of HBs-antigen and Venezuelan equine encephalomyelitis virus. The same method was used for the study of the thin structure of influenza A (H1N1) virus hemagglutinins using monoreceptor antisera.     

Glyco-western blotting: biotinylated dermatan sulfate as a probe for the detection of dermatan sulfate binding proteins using western blotting

Dermatan sulfate was biotinylated through the free amino residue of the core protein. The lysates of mouse and human lung culture cells were electrophoresed and blotted to a nitrocellulose membrane. The membrane was blocked successively with bovine serum albumin, avidin and biotin, and then treated with biotinylated dermatan sulfate followed by visualization using alkaline phosphatase conjugated avidin and its substrates. More than 30 bands were observed on the membrane when 1 microgram/ml of biotinylated dermatan sulfate was used. The binding was prevented by an excess of dermatan sulfate but not chondroitin sulfate A or heparan sulfate. Some of the bands resisted washing with high salt concentration buffer. 60 kDa heat shock protein was found to be a dermatan sulfate binding protein upon two dimensional electrophoresis.     

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.