| 19-nortestosterone (19-nortestosterone,19-NT), is a synthetic steroid hormones, because of its androgen-like effect, it was often illegally used as animal growth promoters. Once the use of the hormones, animal liver, kidneys and other parts would have left the existence of human consumption, which can produce a series of hormone-like effect, undermining the body's hormonal balance, interfering with people's endocrine function, affecting people's reproductive capacity, and potentially carcinogenic to humans. Therefore, this study set up the corresponding ELISA enzyme-linked immunoassay techniques, colloidal gold rapid detection and molecular imprinting technology to ensure the accurate, rapid, high sensitivity detection for 19-nortestosterone.As the 19-nortestosterone is a small molecule, non-immunogenic, 19-nortestosterone can only be immunization antigen and coated antigen after it was coupled with bovine serum albumin (bovine serum albumin, BSA) and ovalbumin (ovalbumin, OVA) respectively. First the 19-nortestosterone was oximated by carboxymethoxylamine and the oximated production (NT-CMO) and then synthesize d 19-NT artificial antigen using anhydride method. Six mice was immunized, and the 6 mice has the best inhibit results, so the 6 mice was selectd to cell fusion, the cell was divided into four 96-well plates, and got hybridoma cell 1A3 and 3B2.3B2 has better inhibit effect, so it was selected to enlarge preparation and purification, the measured affinity constant was 8.42×1010L/mol, and the specificity is very good, almost no reactivity of structural analogues, was identified subtype IgGI type.19-NT-Ab-HRP conjugate was preparaed and identified, enzyme conjugate rate was 0.8797, and antibody titer was 1:12800. A direct competition ELISA detection methods was established, IC50 was 2.9ng/mL, intra-and inter-CV were less than 10%, through the accelerated stability test, predicted that the ELISA kit can stable for at least 9 months at room temperature. After the assessment of specificity, accuracy, stability, and other indicators, the ELISA detection method has a good performance, fully able to meet the need for batch testing.20nm colloidal gold nanoparticles were prepared and were conjugated with 19-NT antibody, the optimized conjugate pH was 8.6, and the optimized conjugate concentration was 7.2μg (antibody)/mL (gold). By optimizing the experiment, the best T-line concentration was 0.3mg/mL, the best C-line concentration was 0.5mg/mL, pH of sample testing should be adjusted in the 7.0～8.0 region, organic solvent content in samples should be controlled at 30% or less, the ionic strength of samples should be controlled in 0.1mol/L below. The detection limit was 5ng/mL for detection of 19-NT standard solution. Through an accelerated stability test, we can predict that the test strip can be stabilized for at least 5 months at room temperature.1mmol 19-NT as the template molecule,4mmol methacrylic acid as functional monomer, 10mmol TRIM as crosslinker, were prepared for 24h at 60℃by precipitation polymerization for molecularly imprinted polymer microspheres (MIPs), which has relatively uniform particle size. The specific surface area was 40.2421 m2/g, the pore size was about 20.3nm. The maximum apparent binding capacity of 94.882μmol/g to 19-NT, the identify factorβvalues was 27.26 for the template molecule which showed the strongest binding capacity and specificity of recognition. Elution of the MIP-SPE conditions were optimized,10% methanol was used as lotion, methanol/acetic acid (9:1, V/V) selected as eluent, when use 4mL eluate, almost all adsorbed 19-NT molecule can be eluted off. At the 11th use of MIP-SPE, the average recovery rate can still reach 85% or more, the entire process of CV values are less than 5%. The linear equation of MIP column for 19-NT was y=0.4188X-4.5569, R2 =0.9983, by optimizing the conditions of use. And MIP has good effects of enrichment, which can multiple 48 times concentration. Therefore,19-NT-MIP can fully meet the solid-phase extraction application requirements, so can be provides fast and efficient means of sample pre-treatment for HPLC, ELISA, dipstick colloidal gold detection technology.
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