Screening Of Monoclonal Antibody Against Recombinant Human Growth Hormone And Development Of Double Antibody Sandwich ELISA For HGH

Human Growth Hormone is a peptide hormone secreted by the anterior pituitary gland in the brain. HGH is a 191-amino acid, single chain 22 kDa polypeptide hormone, it controls so many functions. The growth-promoting action of GH is mediated by IGF-Ⅰwhich is produced mainly in the liver, but also in extra hepatic tissues. GH regulates somatic growth, substrate metabolism and body composition.With the development of hybridoma technique and the progress in enzyme-linked immunosorbent assay, it is possible to establish the better way in measuring hGH in blood serum. ELISA analysis has so many merits in sensitivity,specificity,stability. It is inexpensive and free from radioactive contamination. This sandwich ELISA is an efficient method that can be easily adapted to the automated devices for confirmation and monitoring of hGH deficiency.In this study we immunized rabbits with recombinant human Growth Hormone. Its spleen cells fused with SP2/0 and then cultivated in cell culture plate. Screening the hybridoma cell to get the monoclonal antibodies (McAb). There are two McAbs we obtained named 3E11,2G9. By analysis of titers, Ig subclass, SDS-PAGE, western-blotting, the titers of the serums reach 1:106 and 1:107 by ELISA method, and the McAbs have high purity and specificity.In this study we developed the Double Antibody Sandwich ELISA for hGH. This method using an anti-hGH monoclonal antibody 3E11 as coating antibody to recognize and bind to hGH, the second monoclonal antibody 2G9 as the antibody labeled with the enzyme horseradish peroxidase(HRP), to achieve the detection of hGH. We determined the concentration of the antibody, and their working conditions. According to the value of OD490 standard curve drawn successfully. A double antibody sandwich ELISA assay was established. Detection range is 0.25-20ng/ml, sensitivity of 0.2 ng/ml, the number of concentration has good linear relationship with OD490 values, The intra-assay coefficient of variation (CV) was between 6.56% and 8.7%, while the inter-assay CV was between 7.13% and 9.25%. The average recovery was between 98.06%. These features show that our assay is an efficient method for the determination of hGH. It is expected to develop a growth hormone detection kit through further clinical trials.