Journal of immunoassay最新文献

Highly elevated titers of serum anti-GM1 ganglioside antibodies are closely associated with multifocal motor neuropathy, but low titers are commonly present in normal individuals or other diseases. Current systems for measuring anti-GM1 antibodies utilize the enzyme-linked immunosorbent assay (ELISA), in which serum dilutions are tested for binding to excess antigen immobilized on the surface of microwells. The ELISA system, however, is relatively time consuming, labor intensive, and costly, in addition to being prone to methodological variability. We have developed a novel agglutination assay for the detection of anti-GM1 antibodies, utilizing GM1 ganglioside-coated latex beads. In contrast to the ELISA system, antibody titers may be quantified by testing for agglutination using latex beads coated with decreasing amounts of antigen. The agglutination assay compares favorably to the ELISA system in sensitivity and specificity, but is considerably less costly and takes only a few minutes to perform.

We have devised a protocol for the isolation and identification of a proliferative antigen of the initial cells of wheat stem meristems (termed PAI). We have carried out a variety of immunochemical and immunocytochemical methods, using colloidal gold (CG) complexed with monospecific antibodies to PAI as the marker for the detection of PAI. We have been able to determine the effectiveness of immunoaffinity chromatography in isolating PAI from plant tissues and have shown the advantages of CG over enzyme labels for identification of the antigen. Finally, we have obtained a purified preparation of PAI and have determined its molecular mass (approximately 83 kDa).

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for human osteoclast differentiation factor (ODF/RANKL/ OPGL/TRANCE) utilizing a polyclonal antibody that recognizes both human soluble ODF and mouse ODF in combination with a osteoclasogenesis inhibitory factor (OCIF/OPG) was developed. We can quantify the ODF level in not only human ODF (detection limit: 0.05 ng/ml), but also mouse ODF by virtue of cross-reactivity. Employing this assay system, we demonstrated that ODF is constitutively present as a membrane-bound form in both the human osteosarcoma cell lines, MG-63, HOS and SaOS-2, and the mouse osteoblastic cell line MC3T3-E1.

A very simple and effective procedure which allows simultaneous electroelution of separated proteins from SDS polyacrylamide gel into small quantity of elution buffer is described. Elution parameters have been optimized for maximum possible recovery (50-60%). Protein fractions were collected in physiological buffer and an efficient removal of SDS have been obtained, thus fractions collected were suited for direct testing in cell cultures. Method was used to investigate human T-cell responses to purified secreted M tuberculosis H37Rv proteins. Eight low molecular weight (M.w. range 10 kD to 25 kD) culture filtrate proteins were purified in quantities, sufficient for immunological characterization. Lymphocyte proliferative responses and cytokine release pattern from tuberculosis patients, healthy contacts and healthy controls were studied on stimulation with purified culture filtrate proteins. Immunologically important M.tuberculosis proteins were identified by using this method. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen.

A rapid whole blood culture (WBC) assay system was developed to monitor the inflammatory potential of water samples collected in the Western Cape, South Africa. Water contaminated with inflammatory substances induced the pro-inflammatory hormone interleukin 6 (IL-6). All water samples collected from the Eerste River, Stellenbosch, induced IL-6 secretion, and the quantity of IL-6 secreted is dependent on the concentration and origin of the sample. The lowest IL-6 inducing activity for river water was obtained for samples collected near the origin of the river. Samples at subsequent points downstream showed an increase in IL-6 inducing activity. Drinking water samples collected from selected towns in the Western Cape showed that there were major differences between the inflammatory potential of the water. Of the 15 samples assayed, 7 had low inflammatory activity, 4 had an intermediate inflammatory activity and 4 had high inflammatory activity. The water sources that have a high inflammatory activity may pose a health risk to consumers.

Human tissue prokallikrein is the enzymatically inactive zymogen of a serine proteinase involved in the liberation of vasoactive kinin peptides, and it is supposed that an impaired prokallikrein-to-kallikrein conversion is closely related to certain hypertensive and inflammatory disorders. Progress in understanding the biological role of the proenzyme has been limited by the absence of an accurate assay for the kallikrein precursor. We describe a sandwich enzyme-linked immunosorbent assay to measure human tissue prokallikrein using monospecific anti-peptide antibodies raised against propeptide derivatives. This method could detect a minimum concentration of 60 pg/ml prokallikrein and displayed no cross-reactivity or interference with mature tissue kallikrein. The intra- and inter-assay precision varied from 8-15%, respectively, indicating a reasonable reproducibility of the method. The level of prokallikrein was defined in different human urine samples, and the corresponding dilution curves showed good linearity. The mean recovery of added zymogen was 104%. Prokallikrein immunoassay is the first reported tool for the direct and sensitive quantification of the precursor of tissue kallikrein and should facilitate the precise determination of prokallikrein levels in a variety of biological specimen.

ELISA is a sensitive, specific, reproducible and fast method for detection of antigen-antibody reactions. In case of non-protein antigens as LPS, problems exist, such as poor proportion of coating to microplates, non-specific binding of antibodies to the plastic wells. These problems were resolved partially by Takahashi and co-workers using poly-L-lysine for coating of LPS antigens. To reduce non-specific binding, blocking agent, such as bovine serum albumin (BSA) or casein is commonly used. We have to choose the blocking agent carefully because LPS can bind proteins non-specifically. This process can inhibit binding of LPS-specific antibody to LPS and decrease the sensitivity of method. In this paper we describe an ELISA test for LPS in which normal goat serum is used for blocking. This modification increases the sensitivity of ELISA. This method is useful for detection of LPS (S, R form) and anti-LPS antibody reaction in serological cross-reaction studies.

To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. The optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.

Application of a non-cavitating ultrasonic standing wave to suspended microparticles brings the particles into close approximation and has been used previously to enhance the performance of several diagnostic agglutination tests. The sensitivity of rotavirus detection by ultrasound enhanced latex agglutination was compared with conventional test-card agglutination. Application of ultrasound gave a 32-fold improvement in the sensitivity of detection of rotavirus antigen in buffer compared with the test card method. A novel turbidimetric approach was used to measure agglutination occurring following the test-card procedure (in place of visual examination) and following exposure of commercial rotavirus latex reagents to a 4.5 MHz ultrasonic field (in place of microscopy). The sensitivity enhancement over the conventional method achievable through ultrasonic exposure was comparable whether agglutination measurements were made visually or turbidimetrically and demonstrates the potential for turbidimetry in combination with the ultrasonic method. Turbidimetry offers an alternative to visual assessment that may be more easily incorporated into automated systems.