| Immunolabelling technologies are method for qualitative and quantitative determ ination of the body fluid of the hapten, antigen or antibody. The antigen antibody reac tion was labeled antibody or antigen by using of fluorescein, radioactive isotope, enzy me, ferritin, colloidal gold and chemical (or biological) luminescence agent as tracer. These methods also with the help of fluorescence microscopy, X-ray measuring instru ment, ELISA test, electron microscopy and luminescence immunoassay instrument pr ecision application of various instruments, liquid phase and solid phase immunoassay m-ethod.Time-resolved fluorescence immunoassay (Timed-resolved fluoroimmunoassay, TRFIA) is an advanced sensitive quantitative immunoassay technology. The technolo gy is with lanthanide ions as a marker to mark the antigen or antibody. Because of Ian thanide ions have unique fluorescence properties, the technology can achieve high sig nal-to-noise ratio, thus has high sensitivity, and has the advantages of simple preparati on, markers of storage time, free of radioactive pollution, detection reproducibility, sh ort operating process, the standard curve range is wide, not affected by sample natural fluorescence interference and the application range is very extensive. Time-resolved fluorescence immunoassay compared with enzyme immunoassay, radioimmunoassay has higher clinical application value. Clinical detection of the technology is suitable f or all kinds of antigen and antibody, has been widely used in the market.Testosterone is a hormone, which detection is one important part of clinically six h-ormone detection. Clinically used in testosterone detection reagent mainly are the c he-miluminescence detection reagent which imported and time-resolved testosterone assay kit from testosterone production company PE currently. Testosterone detection reagent and the development of domestic become the focus of our attention. Testoster one as the steroid hormone, is a small molecular hapten molecules in serum testostero ne,98%are present in combination with hormone binding protein, which has brought the difficulty and challenge these particularities to testosterone ELISA R&D work. Therefore, in the development process of testosterone reagent, we will introduce the i dea of heterologous bridge with the development process, structural analogues of test osterone androstene two ketone as the carrier, in its C3introduction containing five m ethylene arm, on the basis of the structure of synthetic NHS active ester, and BSA co-upling by Eu markers thus as a tracer in the reaction system. The indirect competition method was developed for determination of the reagent, serum testosterone levels in plasma.Colloidal gold immune chromatography is a new technology based on ELISA, la-tex agglutination test, monoclonal antibodies and immune colloidal gold labeling tec-hnique, which is marked by colloidal gold, to amplify the response using the antigen antibody reaction specificity. It can be judged by direct observation. This technology has the advantages of simple, rapid, accurate and no pollution. Development of variou-s diagnosis field in clinical medicine detection, hormone detection, detection of food safety, drug residue rapid detection and drug fast [1]. With the development of instant test (POCT) in-depth, colloidal gold reagent has become the main field for the develo pment of POCT at present because of its fast, convenient, with easy to carry, easy to i-ndepth community and remote and a mountainous area, so the requirement of colloid-al gold reagent for detecting acute onset, the harm of diseases related became urgenc-y.This article is composed by three parts.In the first part, four kinds of testosterone and testosterone analogues chemical derivatives and conjugates prepared. In the produce, with testosterone or testosterone analogues-androstene as raw material, then introduced O-(carboxymethyl) hydroxylamine hydrochloride (O-(carboxymethyl) hydroxylamine hemi-hydrochloride) or aminooxy acetic acid hydrobromide (6-Aminooxy-hexanoic acid, hydrobromide) as the arm structure. In the next step, the active ester was synthesized as following:in the NHS and DCC conditions, stirring overnight, the oxime synthesis of NHS corresponding to the active ester structure, use flash’s chromatography to remove unreacted components. Then took active ester and BSA scale with the molar ratio of15:1coupling reaction at room temperature, stirring overnight, the next day to molecular sieve purification, and four types of conjugates concentrations were detected by BCA method. At last, concentrations of testosterone conjugates I, II, III and IV were0.8mg/ml、0.9mg/ml、0.9mg/ml and0.8mg/ml.In the second part, we described the development of testosterone time-resolved fluorescence immunoassay reagent. First, steroid-free serum was prepared with kaolin and activated carbon, and calibrator was prepared with the steroid-free serum just as following:A(0nmol/L)、B (1.0nmol/L)、C(3.2nmol/L)、D(6.8nmol/L)、 E(14nmol/L)and F(51.2nmol/L). The next step is the labelling of testosterone conjugates. Mixed each conjugate1mg with0.5mg Eu3+and incubated for18h at room temperature. The product was purified and detect the testosterone concentration by BCA assay, then the Eu standard solution was added to the purified product and preserved under-20℃.The concentration of testosterone tracers were132μg/ml、135μg/ml、138μg/ml'140μg/ml separately. Step three is the definition of the optimum coating concentration of goat anti-mouse IgG. Detected and compared the fluorescence value of testosterone calibrator A in different coating concentration separately, the optimal goat anti-mouse IgG coating concentration is6μg/ml. Step four is the comparison of four testosterone tracers. Detected the ED50of each testosterone tracer, and chose tracer IV with the lowest ED50as the optimal testosterone tracer. Step five is the definition of sample quantity. Detected the fluorescence value of testosterone calibrator A (Bmax) and the fluorescence value of testosterone calibrator B under different sample quantity separately, calculated the B/Bmax ratio, chose25μl with the lower B/Bmax ratio and the easier operation procedure as the optimal sample amount. Step six is the definition of optimal concentration of testosterone monoclonal antibody and testosterone tracer. The srandard of determination is sensitivity and fluorescence value of testosterone calibrator A, finally the optimal concentration of testosterone monoclonal antibody is6ng/ml, the optimal concentration of testosterone tracer is1μg/ml. In human serum,99%of testosterone is bind with SHBG, so the blocking agent plays important role in the exact detection of testosterone. Step seven is the determination of optimum concentration of blocking agent. Detected the fluorescence level of testosterone calibrator, calculated and compared the B/Bmax of each calibrator in different concentration of blocking agent, evaluated the sensitivity and influence of blocking agent concentration on the reaction system, determined the optimal concentration of blocking agent was30μg/ml. Clarifyed the optimum reaction time in step eight, detected the fluorescence ratio with calibrator A, B, C, D, E,F and Btotal in different reaction time, eventually optimal reaction time is90minutes.Overall evaluation of the reagent was carried out after all the parameters were determined. Analytical sensitivity is0.15nmol/L; The recovery rate of dilution is between98.8%and101.4%, there is a linear relationship between the dilution and concentration. Then analysis the specificity of the reagent, the cross-reactivity of progesterone was0.14%, the cross-reactivity of DHT was0.38%, the cross-reactivity of estradiol was0.07%, the cross-reactivity of17-hydroxyl progesterone was0.02%, and the cross-reactivity of estriol was0.09%, and the cross-reactivity of Danazol was0.12%. In the aspect of precision analysis, the coefficient of variation (CVs) was2.5-6.6%(intraassay)and3.6%～7.8%(interassay). In terms of the evaluation of linear, linear formula is Y=1.453-2.193X, the correlation coefficient r=0.99, R2=1.000,F=7303.334,P<0.001, shows that the linearity of this competition system is perfect; Compared with PE reagent kit,84samples were detected, correlation formula was Y=0.991X-0.331, the correlation coefficient was0.988, R2=0.977, F=3407.808, P<0.001, the result reflected that this reagent can meet the requirements of clinical detection very well.The third part of the thesis mainly introduces the development process of procalcitonin colloidal gold immunochromatography semi-quantitative detection.In the reagent development process, the NC membrane was screened at first, HF135was determined by the comparison of capillary flow, sensitivity, response time and the background; the second step is the definition of the colloidal gold combined pad material, The selection of colloidal gold candidate materials were compared from glass fiber materials, the nonspecific adsorption, colloidal gold constant release, colloidal gold dissolution and release rate, and hemolysis prevention. The final determination is GFCP103000produced by Millipore after a series of experiments; the third step is the screening of raw materials by TRFIA and colloidal gold method. Evaluated29combinations of antibodies successively, contain pairing specificity, sensitivity, color discrimination, and so on. Ultimately,16B5of Hytest was determined as coating material, MJG03of Hangzhou Qi Tai was determined as labeling material. The fourth step is definition of the optimal labeling condition of MJG03, the optimum labeling conditions of MJG03is15μl0.1M K2CO3per milliliter of colloidal gold, and1.5mg MJG03every100ml colloidal gold. The fifth step is to determine the best coating condition of16B5, lOmM PBS (pH7.2) was chosen as the coating buffer by setting different packages. Detection in different coating conditions reagent sensitivity, specificity, T line color degree conditions, choose the best performance of T line. The sixth step is the determination of the concentration of16B5and gold-labeled MJG03, ultimately the concentration of gold-labeled MJG03is OD535=40, the spraying quantity is0.5μl/mm, the concentration of16B5is2.5mg/ml, the spraying quantity is0.12μl/mm, the optimal concentration of goat anti mouse IgG is lmg/ml, the spraying quantity is0.12μl/mm. The seventh step is the establishment of standard, the positive samples of0.5ng/ml,2ng/ml and lOng/ml were measured for20times separately, and then took the average chromaticity as color standard. The evaluation of reagent carried out after related parameters were determined, the sensitivity of reagent is0.2ng/ml, and the reagent did not react with C-reactive protein(10mg/ml), calcitonin(6.4mg/ml), and rheumatoid factor(500IU/ml). The consistency of plasma and serum is perfect. The performance of reagent did not changed under the condition of37℃for21days. The precision of reagent was evaluated by samples with the concentration of0.5ng/ml,2ng/ml and10ng/ml for ten times, and this experiment showed that precision of reagent was perfect. Finally, the reagent was compared with other quantitative reagent, the correlation coefficient is88.9%. |
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