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.
A solid phase method for direct radioimmunoassay in plasma of the new renin inhibitor CGP 60536 has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed a good reproducibility down to plasma concentrations of 0.15 ng/ml (LOQ) with intra- and inter-assay coefficients of variation less than or equal to 20%. The procedure, which requires only small volumes of plasma (25 mu l), is simple to use and well suited for routine analysis. The method allows the investigation of the pharmacokinetics of CGP 60536 in animals and man given low oral doses of the drug.
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.
To assess mice interleukin-12 (IL-12)-secreting cells at a single cell level, we have developed a murine IL-12 specific enzyme-linked immunospot (ELISPOT) assay. The application of the newly developed method clearly showed the frequency of IL-12-secreting cells in the resident peritoneal exuded cells was higher than other organs of normal DBA/1J mouse. Moreover, we determined the frequency of IL-12-secreting cells in the spleens of five strains of inbred mice, and found the incidence of IL-12 secretors in the strain C57BL/6 to be greatest, and significantly greater than four of the others. These results are compatible with the predicted evidence, supporting this ELISPOT assay for IL-12-secreting cells is accurate. The procedure provides a useful tool for investigating complicated immune responses at a single cell level.
The Lyme immunoblot uses recombinant proteins on the blot so it is more sensitive than the western blot. This test can detect Lyme disease as early as one week after exposure to the Borrelia bacteria and detect late-stage Lyme when antibodies are typically low. Like the western blot, an immunoblot result has to meet certain criteria for a positive result. Additional species of Borrelia bacteria fall into the Tick-Borne Relapsing Fever Group (TBRF). The TBRF group causes symptoms similar to Lyme disease
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.
(2000). Multiparameter Flow Cytometry. Journal of Immunoassay: Vol. 21, No. 2-3, pp. 255-272.
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.
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.
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.
Immunohistochemistry is a very versatile immunopathological tool for the study of distribution and differentiation of antigens and of the presence of in vivo-bound immune complexes. In addition, these methods are invaluable for detection of circulating antibodies to the various antigens. Such methods may be the only ones of choice in certain situations. For the detection and quantitation of these antigens, it is very essential that the immunohistochemical methods for detecting them are properly standardized, with the inclusion of appropriate controls.
Immunological recognition is mediated by T and B lymphocytes and by immunoglobulin molecules secreted by plasmocytes. The T cells recognize protein antigens by means of their T-cell receptors (TCRs) after the antigen has been processed into peptide fragments, a topic discussed in Chapter 13. The B cells recognize antigens by means of B-cell immunoglobulin receptors, which are actually antibody molecules anchored in the B-cell membrane. Unlike T-cell receptors, B-cell receptors are able to recognize the native tertiary structure of a protein antigen. The antibody molecules that are subsequently released by the B cell, after its differentiation into a plasmocyte. possess the same specificity as the receptors of the triggered B cell; therefore, they are also able to recognize the native conformation of the protein antigen. The first selective theory of antibody diversity was developed at the beginning of the century by Paul Ehrlich, who proposed his “side chain” theory to explain the appearance of specific antibodies. According to this theory, lymphocytes possess on their surface a variety of side chain groups or receptors that are able to combine in a specific manner with different antigens. The interaction of the antigen with one of the side chains was believed to result in the release of that side chain group from the cell surface and to trigger the subsequent synthesis and release of large numbers of the same side chains. According to Ehrlich, the antigen selects, on the surface of the lymphocyte, a receptor with a complementary shape, and it induces the cells to excrete large numbers of this receptor in the form of specific antibodies. This side chain theory is remarkably modern, since it is similar to the currently accepted clonal selection mechanism, which links the specificity of antibodies appearing in the serum with the presence of preexisting clones of cells possessing the same antibodies immobilized at the cell surface. The molecular mechanisms of antigen recognition by an antibody anchored in the B-cell membrane or by a free antibody molecule are the same. However, it is much easier to study the binding of antigens to free antibodies than to antibodies attached to B cells, and most of our knowledge concerning antigen-antibody binding has been derived from studies with free antibody molecules. The term antigen refers to any entity that is able to generate an immune response in higher vertebrates and to be recognized by the products of the immune response. The ability of antigens to react specifically with complementary antibodies is known as antigenic reactivity, and their
Antibody generated from ractopamine-hemiglutarate-KLH was used to develop a ractopamine ELISA. The antibody showed good sensitivity in phosphate buffer, with an IC50 of 4.2 ng/ml (ppb) toward ractopamine and 16.2 ng/ml toward glucuronides of ractopamine conjugated to the phenethanolamine phenol of ractopamine. Phenylbutylamine phenol glucuronides of the (RS, SR) ractopamine diastereoisomers showed about 4% cross-reactivity, but the glucuronide of the (RR, SS) diastereoisomers conjugated at the same phenolic group showed no detectable reactivity with the antibody. The antibody generally had cross-reactivity towards compounds with bis-phenylalkyl amine structures rather than compounds with simple branched N-alkyl substituents. For example, the antibody showed little or no cross reactivity towards clenbuterol, isoproterenol, metaproterenol, and salbutamol, but cross-reacted with dobutamine. The system demonstrated a matrix effect similar to other enzyme immunoassays, dilution of urine decreased but did not eliminate the matrix effect.
Immune precipitation is the formation of insoluble complexes as a result of the specific interactions between antigen molecules and the corresponding antibody molecules, both in aqueous solution. Usually, the largest amounts of precipitate are obtained when soluble antigens and antibodies are present in approximately equal concentrations. Therefore, to determine the concentration, or just the presence of antibody, precipitation requires considerable amounts of antigen; the method usually does not allow the detection of less than microgram quantities of antibody; that is, it is about loo0 times less sensitive than agglutination. Contrary to agglutination, divalent IgG (and not decavalent IgM) is the immunoglobulin with the strongest precipitating power. A major breakthrough that caused the diversification of immune precipitation into a variety of different and powerful analytical methods was the development of precipitation in gels. This approach gave rise to methods permitting, for example, the distinction of small differences between antigenic sites, and the characterization of 100 or more different blood serum proteins.
One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazidehorseradish peroxidase (testosterone-3-O-CMO-ADH-HR-P) was used as tracer. To the testosterone antibody coated micro-titer wells, standard or serum samples (100 muL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 muL) were incubated for 1 h at 37degreesC. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C-18, C-19, C-21, and C-27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intraassay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r =.98 (n = 100).
