OBJECTIVE Generation of specific monoclonal antibodies (mAbs) against 20K and 22K human growth hormone (hGH) and development of ultrasensitive immunoassays to quantify 20K and 22K hGH. METHODS Mice were immunized with recombinant 20K or 22K hGH. Hybridoma cells were screened with biotinylated 20K and 22K hGH simultaneously. The specific mAbs were further characterized and used for construction of isoform specific assays. The ultrasensitive chemiluminescent assays were developed with AMDEX streptavidin-HRP and a sensitive substrate. RESULTS The 20K hGH specific mAb 1G12 and the 22K hGH specific mAb 5E1 showed less than 0.1% cross-reactivity to 22K or 20K hGH by competitive binding assay, respectively. Western blot analysis also confirmed the specificity of mAb 1G12 and mAb 5E1. Using mAb 1G12 and mAb 5E1, 20K and 22K specific assays with working range of 2-2000 pg/mL were constructed. The 22K hGH concentrations in 103 serum samples from different healthy subjects in the basal GH state were 343.7+/-421.5 pg/mL (18.6-1820 pg/mL). The 20K hGH concentrations were 30.7+/-37.5 pg/mL (2.4-205pg/mL). The ratios of 20K to 20K plus 22K hGH were 9.8+/-4.4% (3.3-28.3%). Both 22K hGH and 20K hGH concentrations in women (465.9+/-476.3 pg/mL and 43.7+/-46.1 pg/mL, n=47) were significantly higher than those (241.1+/-337.0 pg/mL and 20K hGH 19.8+/-23.0 pg/mL, n=56, P<0.01) in men. However, there was no difference in the proportion of 20K to 20K plus 22K between men and women (P>0.05). The strong correlation between 20K and 22K hGH (R=0.914, P<0.01) indicated the constant proportion between 20K and 22K hGH in the basal GH state of healthy subjects. CONCLUSIONS Specific monoclonal antibodies and ultrasensitive chemiluminescent immunoassays for 20K and 22K hGH were generated. The ultrasensitive immunoassays are essential for the determination of 20K and 22K hGH in the basal GH state. This universal ultrasensitive immunoassay form can be adapted to other immunoassays for broad application.