Development Of A One Step Strip For Swine Early Pregnancy Diagnosis

Progesterone (P4) is a kind of steroid hormone, which is mainly produced by the corpus luteum and placenta. The P4level was changed regularly during the sexual cycle of female animals. P4keeps a lowest level in estrus and a higher level during luteal phase, still higher during pregnancy phase. Traditionally, P4was detected by radioimmunoassay(RIA), because of its expensive equipment and radiation pollution of the environment, it is unfit to use in the production. Based on antigen-antibody binding, colloidal gold test strip is used to provide diagnosis of early pregnancy pig in this paper. The test strip does not require professionals to use, easy to promote in the market. Colloidal gold test strip in this study for swine early pregnancy diagnosis is made through synthesizing artificial antigens of progesterone and monoclonal antibodies.1Development of Complete Antigen of ProgesteroneThe purpose of the study was to synthesize the antigens of P4. The arboxymethyloxy method was adopted to synthesize progesterone derivative by using O-Carboxymethoxylamine hemihydrochloride. The incomplete antigens were identified by the thin layer chromatography (TLC) and LC/MS. P4derivative was conjugated with carrier proteins BSA and OVA through Mixture anhydrides method for animal immunization and ELISA tests. The conjugates were confirmed by UV spectrophotometer and SDS-PAGE. The results showed that the mobility ratio of P4and the synthetics were0.4and0.2, respectively. This implied that the oxime was successfully produced. The results of LC/MS showed that the molecular ion peak of P4-3-Carboxymethoxylamine hemihydrochloride was386.2, which was to accord with the theory molecular. Ultraviolet all wave scan showed that the maximum absorption wavelength of BSA was278nm, while the maximum absorption wavelength of P-BSA became255nm. In addition, the maximum absorption wavelength of P-OVA was255nm, while the maximum absorption wavelength of OVA became278nm, this implied that the artificial antigen was successfully produced.The conjugates were confirmed by UV spectrophotometer and SDS-PAGE. Their molar ratios of hapten and the carrier protein were30:1and4:1. Then immune New Zealand rabbits with P-BSA, and obtain P polyclonal antibody after4times immunity. The rabbit serum antibody effective value was determined with indirect ELISA and the specificity of the rabbit serum antibody was determined by indirect competitive ELISA. The results show that the rabbits serum antibody was determined as1:219, its competitive inhibition (IC50) was207ng/mL. Ultimately it is proved that complete antigen can stimulate animals to produce high titer and specific anti-serum against progesterone. Hapten progesterone-3-(O-carboxymethyl) oxime and artificial antigen P-BSA, P-OVA were successfully synthesized.2Development of Monoclonal Antibody (McAb) against ProgesteroneTo prepare a monoclonal antibody, after immunize BALB/c mice with P-BSA for4times, the spleen cells of the immunized mice were taken out and fused with SP2/0cell in PEG1450. One hybridoma cell lines designated as2C11, which can produce monoclonal antibody(McAb) against P4, were screened with hybridoma technique. The indirect ELISA titer of ascites was1:204800. And the OD450value of IgG1and Kappa were0.481and0.601, respecticely. The other OD450value of subtypes were less than0.2(if OD450≥0.2, it was positive.). This results suggested that the antibody subtype IgG1was secreted by the McAb, and the light chain type was Kappa. Furthermore, the OD450average means in the supernatant were1.10,1.12,1.11,1.12and1.19, respectively, after subculture and many times resuscitation from storing frozen. This result suggested a stable antibody-secretion.3Development of A One Step Strip for Progesterone DiagnosisColloidal gold test strips for progesterone was prepared by purified monoclonal antibody ascites from2C11hybridoma cell. Anti-progesterone monoclonal antibody, secreted by2C11, and nanocolloidal gold particles were coated in the gold conjugate pad based on the immune competition method and the concentration of the McAb is18μg/mL. P-OVA was coated on the surface of nitrocellulose filter membrane (NC) as the test line (T line) and the anti-anti-body (goat anti-mouse IgG antibody, targeting at the P4monoclonal antibody) was coated on the surface of NC as the control line(C line), and the the concentration of P-OVA and goat anti-mouse IgG antibody were0.548mg/mL and0.625mg/mL. P-BSA antigen and progesterone was combined with progesterone antibody by immune competition reaction. Positive or negative judgments were based on whether T-line displayed rosy red. The whole detection process could be performed in10minutes. Detection limit can reach100ng/mL.No cross-reaction was observed to testosteron and estradiol. The method showed high sensitivity and specification, which could be efficiently used for pregnancy diagnosis in sows.