Development of Isotopic and Non‐Isotopic Microwell Based Immunoassays for hCG Using¹²⁵I and Biotin Labeled hCG

Abstract

Isotopic and non-isotopic immunoassays of hCG, based on the principle of competitive inhibition, using micro-well as solid support and 125I and biotin as labels for hCG, have been developed. In both the assays, rabbit polyclonal antibody was immobilized onto micro-wells. In the non-isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of biotinylated hCG were incubated for 1 hour at 37 degrees C. After incubation, wells were washed and 100 microL of streptavidin-HRP conjugate was added to each well and incubated again for a half hour at 37 degrees C. Bound enzyme activity was measured using tertramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. In the isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of 125I-hCG were incubated for 1 hour at 37 degrees C. The bound radioactivity was measured using a gamma counter. The sensitivities of the non-isotopic and isotopic assays were 0.12 IU/mL and 0.1 IU/mL, respectively. The intra- and inter-assay CVs for both the assays were less than 12.3%. There was a good correlation between the developed non-isotopic and isotopic immunoassays (r = 0.97, n = 20).

Toward a Fast, Easy, and Versatile Immobilization of Biomolecules into Carbon Nanotube/Polysulfone-Based Biosensors for the Detection of hCG Hormone

“…[39][40][41] The detection limit of the sensor was determined to be 14.6 mIU/mL (defined as twice the standard deviation of the blank solution) obtained from the linear regression of the inset in Figure 7 (current ) 17.28 -0.0046[hCG]). It is comparable with previous works 42,43 and with commercial ELISA kits. 37 Despite that this detection limit is higher than other reported heterogeneous immunoassays, [44][45][46] the potential of our immunosensor is not diminished since the detection of the pathologies of interest (ectopic pregnancy, trophoblastic and testicular cancers) are found at higher concentrations.…”

Asp330 and Tyr331 in the C-terminal Cysteine-rich Region of the Luteinizing Hormone Receptor Are Key Residues in Hormone-induced Receptor Activation

“…24 h post-transfection, cells were detached using trypsin/EDTA, and transferred to white Clear TC-treated plates (Greiner Bio-one Alphen a/d Rijn, The Netherlands), 48 h post-transfection, medium was replaced by serum-free culture medium supplemented with 0.1% bovine serum albumin and 25 mM HEPES and a 2-fold dilution series of LH, hCG, Org 41841, or Org 42599. For mass to mole conversion for LH and hCG, molecular masses of 29.5 and 36.7 kDa were assumed, respectively (24,25). After 6 h stimulation, wells were aspired and cells were lysed with 25 mM Tris phosphate, pH 7.8, 8 mM MgCl, 1 mM dithiothreitol, 15% glycerol, 1% Triton X-100 and analyzed for luciferase (cAMP-responsive element driven) and Renilla luciferase (transfection control) activities using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega Corporation, Madison, WI).…”

DNA-Redox Cation Interaction Improves the Sensitivity of an Electrochemical Immunosensor for Protein Detection

“…Recently, immunoassays based on various signaling techniques, such as radioimmunoassay [ 9 ], colorimetric assay [ 10 ], fluorescence immunoassay [ 11 ], and electrochemical assay [ 12 ], have been developed to merge the benefits of different approaches [ 13 ]. Among those, electrochemical immunosensors exhibit the merits of cost effectiveness, good portability, and excellent sensitivity [ 14 ].…”