Establishing And Applying Of ELISA Testing System Of Porcine Growth Hormone

Porcine growth hormone(pGH) is also named after porcine somatotropin(pST),pGH is a 191-amino acid produced and stored in specific cells of the anterior pituitary gland,pGH has profound effects on tissue growth and metabolism。It can enchance the uptakes of glucose and amino acid,the synthesis of protein and fatty decompose,also promote the speed of growth,the ratio feed uptake and lean muscle.With the development of hybridoma technique and the success of the monoclonal antibody against pGH,it is possible to detect the level of pGH in blood.Enzyme linked immunosorbent assay(ELISA) is think highly of a method,it has many virtues,such as non-radiation pollution, strongly specificity,high sensitivity and timing-shorted.It is not demanded for laboratory condition.Constructing the test of ELISA,it played a role in the foster and reproduction of porcine and genetic transmission and expression.The study taked an action on endocrinologist.Anti-serum was produced by immunizing rabbits with porcine growth hormone.Their serum was purified through SAS.Then the conment protein(21mg/mL) was tested through ultraviolet spectrophotometer.BALB/c mice were immunized with porcine growth hormone(pGH).The immunized spleen cells fused with SP2/0 and then inculated in cell culture plate.Monoclonal antibodies(McAb) were obstained by hybridoma technique.Three hybridoma cell strains secreting McAbs for porcine growth hormone were detected by ELISA.They were named by 1B₄, 1C₄ and 2C₅ separately.There were three McAbs(1A₃,1A₅ and 1B₅) in our Laboratory.By analysis of titer,affinity,Ig subclass and antigen recognizing site,the 1A₃ was the best McAb.PGH ELISA is an enzymatically amplified "two-step" sandwich-type immunoassay.In the assay,Standards,controls and unknow serum samples are incubated in microtitration wells which have been coated with anti-pGH capture antibody(1B₄) incubated.After incubation and washing the wells are treated with another anti-pGH detection(anti-serum) antibody.Anti-rabbit of antibody labeled with the enzyme horseradish peroxidase(HRP) was the second antibody.After a second incubation and washing step,the wells are incubated with the substrate OPD.An acidic stopping solution is then added and the degrer of enzymatic turnover of the sunstrate is determined by single wavelength absorbance measurement at 490 nm.The concentration logarithm takes on linear correlation with OD_490nm at the range of pGH content from 20ng/mL～2.0μg/mL,the lowest detectable limit(LDL) is 50ng/mL.The content of unkown sample is computed comparied with standards.This assay method is simple and sensitive.Contents of hormone in Songliao black swine blood which was detected in practical application was 0.601±0.012μg/mL,0.801±0.014μg/mL,0.924±0.022μg/mL.Contents of chondrotropic hormone in swine blood at the aged of 180 days consistent with it in swine blood at the same age which reported in our country.BLAB/c mice were immunized with pure porcine growth hormone(pGH),and hybridoma cell strain secreting McAbs for porcine growth hormone were obstained.To construct a mean of testing the level of growth hormone in porcine blood and tissue,this method has quick,sensitive and specific advantage.It provided some informance in species selection,and prepared some theorial and material foundation for animal breed and secretory mechanism of the frequence of pGH.