Development Of A Chemiluminescence Immunoassay For Prolactin

Objective: McAb of proclatin was labeled with horseradish peroxidase(HRP) heptaiodic acid sodium by using as a coupling reagent. Find out substrate which to suit chemiluminescence enzyme linked immunoassay for determining prolactin. We developed and optimized chemiluminescence enzyme linked immunoassay for determining prolactin by using McAb to labeled. Final, developed chemiluminescence enzyme linked immunoassay for determining prolactin.Method: (1) Find out substrate and optimized environment in which working through to compare three substrate whose light quantity and stationary phases. (2) Developed and optimized chemiluminescence enzyme linked immunoassay for determining prolactin. (3) Research of level of the system which chemiluminescence enzyme linked immunoassay for determining prolactin.Results:(1) Compared three substrate which was used CLEIA, through research of light quantity and stationary phases. Then it was used substrate, which chemiluminescent ultra sensitive HRP microwell substrate of BioFX. And the best concentration of substrate was definited, which is dilution of 10 fold. There was a fine correlation between defferent concentrations and light quantity of them. Besides the substrate have a stationary phases which keep 60 minutes. Therefore I considered the substrate can satisfy requisition which range and mass sample of the detection.(2) McAb of proclatin described above was labeled with horseradish peroxidase(HRP) heptaiodic acid sodium by using as a coupling reagent. Then McAb of proclatin conjuncted microwell plate through mean of adsorption, and labeled McAb of proclatin was used detection. To utilize system of detection by using chemiluminescence, developed chemiluminescence enzyme linked immunoassay for determining prolactin. The optimal package concentration of McAb of proclatin was 1μg/mL, solution of citrate was buffer, and its time was 24h at 4℃. Blocking buffer was 1% bovine serum albumin, closed for 24h at 4℃. Optimal dilution titer of horseradish peroxidase labeled McAb of proclatin was 1:500. Proclatin was diluted with blood serum of horse, and it is not contrast that standard samples have different concentration compared with government standard, meanwhile they had a good parallelism. In detection, quantity of standasd sample or blood serum was 50μL, and labeled McAb was 50μL too. And when horseradish peroxidase labeled McAb of proclatin was working, the temperature was 37℃, kept 60 minutes. The method of detect proclatin spend two hours. (3) The research get possessory composition of process which detection for proclatin and establish method of detection. Then the sensitivity, specificity, precision, recovery rate and stability of the method were detected. The results showed that the preservation of the kit was one year at 4-8℃, and high specificity, sensitivity, pricision of the kit were determined. And it is a simple and easy, accurate and fast method for detecting proclatin in sample. The comparison between the method and radioimmunoassay or introduced method is not contrast.