Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems.

Because early detection is the first step in successful therapy, increasing the sensitivity of detection systems has always been considered as one of the major trends in development of these technologies. Therefore, we have fabricated a high-performance poly-horseradish peroxidase (HRP) complex and analyzed it in different formats of immunodetection systems. To construct this complex, dextran-aldehyde was prepared by oxidation of dextran in the presence of sodium periodate. Activated polymer was then coupled to lysine amino acids and accomplishment of the process was evaluated with trinitrobenzenesulfonic acid. Following conjugation of HRP to free amino groups of lysine, the stage's accuracy and the rate of conjugation were demonstrated by SDS-PAGE. Then, conjugation of poly-HRP complex to streptavidin by biotin was performed. The results of a series of experiments confirmed the complete synthesis of streptavidin-poly-HRP complex by this procedure. Finally, we compared our harvested complex with the golden standard complex available for ELISA and immunohistochemistry (IHC). The results showed the high efficiency of the synthesized complex. Consequently, this complex can be applicable in highly sensitive detection technologies. Conjugating this complex to any antibody by using biotin-streptavidin bridging and preparing poly-HRP-labeled antibodies will be a valuable multifold approach to increase the sensitivity of detection systems, which can be applicable in ELISA, immunocytochemistry, and IHC methods.

Investigation in vitro Expression of CatSper Sub Fragment followed by Production of Polyclonal Antibody: Potential Candidate for The Next Generation of Non Hormonal Contraceptive.

OBJECTIVE: CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper (1-4) ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. MATERIALS AND METHODS: In this experimental study, a small extracellular fragment of CatSper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1) pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2) The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) and western- blot, and 3) The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. RESULTS: The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with (p<0.05) unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CONCLUSION: CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive.

Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.