Development Of The Colloidal Gold Immuno-Chromatographyic Strips For The Early Pregnancy Diagnosis Of Hu Sheep

The amount of progesterone in blood during early pregnancy is much higher than that during estrus. And this phenomenon could be used for early pregnancy diagnosis. The traditional detection methods, such as radio immunoassay (RIA), enzyme immunoassay (EIA), latex agglutination inhibition test (LAIT), have not been applied widely because of the requirements for complex equipment and professionally operation. At the same time, the Gold Immune-Chromatographic Assay (GICA) is a new immunological detection technology since the early 1990s. This technology is convenient and quick, whose results could be observed by eyes, so that it has been applied widely in food safety and hormone detection.In this study, Strips for Semi-quantitative detection of Progesterone were worked out under the competition principles using GICA. After the HU sheep had made mating for 21 days, we measured the amount of progesterone in their blood, and then we compared the amount with critical value for early pregnancy diagnosis. The test results were listed as follows:1. The blood samples were collected from 85 random selected HU sheep inseminated after 21 days,then testing the concentration of blood progesterone. The results showed that, the concentration of blood progesterone about pregnant sheep was 2.33±0.70ng/mL,and the unpregnant was 1.24±0.33ng/mL,the difference was significant.The early pregnancy diagnosis critical value was above or equal to 1.7ng/mL with pregnancy,below 1.5ng/mL with unpregnancy, the critical value between 1.7ng/mL and 1.5ng/mL with dubious pregnancy. According to the above standards, the definite pregnancy diagnosis rate was 100%, definite unpregnancy diagnosis rate was 82.61%.2. The colloidal gold particles of 30 nm was prepared by the tri-sodium citrate reduction method,and then identified by two different ways.The key experiment conditions to develop gold-monoclonal antibody of progesterone were optimized.The spheroidal colloid gold particles of 30 nm was shown in color of bright red,and the equal particle ultrastructure was observed through transmission electron microscope(TEM); The minimal stable concentration (MSC) of gold-monoclonal antibody was 16μg/mL,and the suitable stable concentration (SSC) was 19.2μg/mL.The pH 8.5 was appropriate.Obvious low electron density halo was obversed around the particle of gold-monoclonal antibody complex,and the OD value of 520nm was in the range of 0.2 to 0.4,thereby preparing for further research on the gold immunochromatography test strip of rapid diagnosis of pregnancy.3. Strips for Semi-quantitative determination of Progesterone were prepared with the competitive principle of Gold Immune-chromatographic Assay(GICA),and developed with the conjugate pad made of gold-progesterone monoclonal antibody complexes, while with the lla-Hydroxy Progesterone Hemisuccinate:BSA and goat anti-mouse IgG as test line and control line.The materials of sample pad,conjugate pad,NC membrane were domestic SB-08 glass fiber membrane,import Ahlstrom 8964,Millipore135.The optimal concentrations of 11a-Hydroxy Progesterone Hemisuccinate:BSA and goat anti-mouse IgG was 1.6mg/mL and 1.2mg/mL,and gold-progesterone monoclonal antibody was 1:2.NC membrane was closed with 3% BSA. The specificity, stability and stability of the assay were excellent.27 sheep were detected with the Strips,the results showed that the Coincidence between the strip and RIA was 77.8%,the Coincidence between the strip and production record was 77.8%,66.7%.