Development And Application Of Immunoassays For Detection Of 19-Nortestosterone

19–Nortestosterone (NT) is often used in veterinary as well as in human medicine for the treatment of protein deficiency diseases and osteoporosis. This anabolic androgenic steroid has also been employed as a doping agent to boost muscular strength and performance in sports and horse racing, and as a growth–promoting agent to accelerate weight gain and improve feeding efficiency in animals. But NT and it metabolite residues damage liver functions and cardiovascular system, cause endocrine dyscrasia, cerebral dysfunction, emotional instability and other side–effects. Therefore, it is forbidden in most countries, except for medical treatment and scientific research.Traditionally, NT residue analysis has relied upon gas chromatography coupled to mass spectrometry (GC–MS) or liquid chromatography coupled to mass spectrometry (LC–MS), and other classical analytical methods. Chromatographic techniques provide sensitive and confirmatory analysis, however, they usually require highly skilled personnel and expensive equipments. Moreover, these laborious sample pretreatment procedures involve numerous extraction steps that are time–consuming and unsuitable for routine analysis of a large number of samples or on–site determinations. Compared with instrumental methods, immunoassays are portability and cost–effective, with adequate sensitivity, high selectivity, and simple sample extraction process. Therefore, immunochemical techniques become a popular and are increasingly considered as alternative or complementary methods for residue analysis nowadays.The objective of this study was to develop a rapid immunoassay for detection of NT residue in animal–origin products, including the synthesis and identification of artificial antigen, preparation and characterization of NT polyclonal antibody and monoclonal antibodies, NT–kits development and optimization, the preparation of immunological gold–labled strip, and the development of GC–MS analysis.1. Succinic anhydride method was employed to prepare NT–17–(succinic anhydride) ester, which was proved by (+)ESI–MS spectrum and 1H–NMR spectrogram. The 17β–19–NT hapten was conjugated to BSA as immunogen with the EDC method, and to OVA as detection antigen by mixed–anhydride technology respectively. The results of infrared spectra (IR) and UV–visible spectra indicated that the artificial antigen was synthesized successfully and the conjugation ratio of NT–BSA was 18:1. Balb/C mice immunized with NT–BSA were introduced to obtain NT pAb, whose antibody titer for indirect ELISA was 1:25600, and IC50 value was 27.3 ng/mL by icELISA. The antisera cross–reacted withα–NT about 69%, but not higher than 0.05% to other steroids tested. The results showed that the artificial antigen was synthesized successfully, which lay a solid foundation for preparing NT–Kits and a rapid test strip.2. An indirect heterologous competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody for analysis of NT in animal urines was developed. This assay was sensitive and had a linear range from 0.06 to 31.2 ng/mL, with IC50 and LOD values of 1.55 ng/mL and 0.04 ng/mL, respectively. After optimization, the best parameters for pH value and phosphate ion concentration were determined to be 7.4 and 10 mM in assay buffer. It also indicated that the concentrations of methanol and NaOH in dilute solution should not be higher than 30% and 10%. Except for cross–reactivity (CR) toward trenbolone (9.6%) and estradiol (6.4%), no significant CR was observed for other analogues tested. To reduce the matrix interference, 1:20, 1:20, 1:20 and 1:50 dilution folds were chosen for pork, beef, dog and horse urine, respectively. The recoveries of NT spiked in the four matrix were in the range of 85 ~ 110%, with RSD values from 4.4% to 16.3%. when stored at 4℃, the NT–Kits could preserve 6 months or longer, and the program could save 30 ~ 90 min after the incubation process was optimized.3. Five hybridoma cell lines named NT–1, NT–2, NT–3, NT–4 and NT–5 were screened out through cell fusion technology, and the corresponding mAbs were of the IgG1 isotype with a k light chain, and the affinity constants (Ka) of all mAbs were between 2.6 and 4.7×109 L/mol. The titer and IC50 values of purified ascites were in the range of 0.64 ~ 2.56×105 and 0.55 ~ 1.0 ng/mL respectively. Based on the NT–1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT, which dynamic range was from 0.004 to 85.8 ng/mL, with a LOD and IC50 values of 0.002 ng/mL and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) toα–NT, negligible cross–reactivity to other compounds was observed. The correlation coefficient between the established icELISA and LC–MS/MS method was excellent (R2 = 0.9871).4. Based on the hybridoma of 3B8–E6, an heterologous direct competitive ELISA (dcELISA) method to detect NT residue was developed. Under the optimal conditions, this assay exhibited an working range of 0.004 ~ 19 ng/mL, with IC50 and LOD values of 0.28 and 0.002 ng/mL, respectively. Except for a minor cross–reactivity withβ–boldenone (6.9%) and trenbolone (1.2%), the other interference to this assay was negligible (<0.05%). The dcELISA can tolerate higher concentrations of methanol (30%), while the best contents of Tween–20 was determined to be 0.05%, and acetonitrile and acetone should not be higher than 10% in assay buffer. The stabilization studies showed that the dcELISA kits can be stored for at least 180 and 240 days, at 4℃a nd–20℃, respectively. After three sample pretreatment procedures checked, simple dilution method was recommended for further use. When applied to bovine samples, the correlation coefficients (R2) of ELISA and GC–MS data were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney.5. This chapter presents the generation of colloidal gold–based strip to detect NT residue in animal tissues. Colloidal gold particles with the diameter of 15 nm were prepared by the trisodium citrate reduction method, colloidal gold labelled NT mAb as a detection agent was conjugated under pH 9.0, and the immunochromatographic strip was assembled according to the competition theory. The visual sensitivity of strip was 1.6 ng/mL, therefore the detection limit was determined to be 8 ng/mL after 5–fold sample dilution. Of all the analogues, this assay exhibited cross–reactivities to trenbolone (15.6%) andβ–boldenone (7.8%), but negligible CR values (<0.05%) to other chemicals tested. Under 4℃and room temperature, the strip can be validitied for at least 12 and 6 months, respectively.6. A GC–MS method for determination of NT residues has been developed. Animal tissues were hydrolyzed in an acetate buffer, and the homogenate was extracted with methanol, then the fat was deprived with n–hexane. The analytes were subjected to a SPE C18 cartridge for clean–up, and the dried extracts were derivatized with heptafluorobutyric anhydride (HFBA). Using electron impact mass spectrometry (EI–MS) with positive chemical ionization (PCI), four diagnostic ions (m/z 666, 453, 318 and 306) were determined. An equation of linear regression with Y=– 68 354 + 467 084X (R2 = 0.9997) was obtained, over the concentration of 1 ~ 20 ng/g for NT. When applied to spiked samples in animals, the recoveries ranged from 63% to 101%, with CV values of 2.7% ~ 8.9%.