Immunocolloidal technique for the detection of estradiol by immunocolloidal gold test paper
ã€Key words】 Estradiol Estradiol (17β-Estradiol) is an important component of natural estrogen. In order to prevent precocious puberty in children, the detection of estradiol in foods, especially livestock and poultry products, cannot be ignored. Colloidal gold immunochromatography (GICA) is gaining more and more attention in the detection of small molecule organic matter. The basic principle is that colloidal gold particles can stably adsorb proteins, and the biological activity of the protein has no obvious change, so it can replace the enzyme-labeled antibody [1, 2]. In this experiment, colloidal gold particles labeled with estradiol monoclonal antibody were combined with the corresponding estradiol conjugate immobilized on the nitrocellulose membrane, and the color of the estradiol conjugate was fixed at the detection line. [3, 4]. The method has the advantages of high sensitivity, strong specificity, simple and rapid, low cost and easy interpretation, and is suitable for preliminary screening of samples. First, materials and methods 1. Reagents and instruments chloroauric acid (Shanghai Chemical Reagent Factory); bovine serum albumin, egg white albumin, rabbit anti-mouse polyclonal antibody (Tianjin Lianxing Biotechnology Co., Ltd.); estradiol standard, estradiol monoclonal antibody, 17β -Estradiol-6-one6-(o-carboxymethvyoxime) Sigma, USA; other reagents are domestically analyzed. Nitrocellulose membrane, glass fiber membrane (Millipore, USA); Beckman Du530 spectrophotometer (Beckman, Germany); cryogenic high-speed centrifuge (military medical academy); spotting machine (Bio-Dot, USA). 2, method (1) Preparation of colloidal gold Dissolve chloroauric acid in tri-distilled water to a final concentration of 01 g/L. First, carry out a boiling water bath. After boiling the chloroauric acid solution, add 1% trisodium citrate 25 ml per 100 ml. Stir rapidly in a boiling water bath until the color of the chloroauric acid solution was stable. Continue to boil the water bath for 10 min [5]. After cooling to room temperature, the particle uniformity and particle size were measured by transmission electron microscopy and spectrophotometer. Finally, it was stored in a refrigerator at 4 ° C for use. (2) Determination of the optimum amount of labeled protein The colloidal gold solution was adjusted to pH 82 with 02 mol/L K2CO3, and then 9 tubes were taken, and 10 ml of colloidal gold solution was separately added. After the estradiol antibody was diluted stepwise, each equal volume of the diluent was sequentially added to the above test tube, mixed, and a control tube without an antibody was additionally provided. After standing for 10 min, add 01 ml of 10% NaCl to each tube, mix and let stand for 2 h. Observe the change of colloidal gold solution in each tube. The color of the solution without added protein and insufficient addition is changed from red to blue, and the amount of added protein is reached. The solution at or above will remain unchanged. The amount of labeled protein is 20% based on the amount of protein that does not change color in the lowest stable colloidal gold solution. (3) Labeling of colloidal gold probe The antibody was dialyzed against 0005 mol NaCl solution overnight, and the protein precipitate was removed by centrifugation to adjust to 0.05 mg/ml. 100 ml of colloidal gold was taken, and the colloidal gold solution was adjusted to pH 90 with 0,2 mol/L K2CO3. Slowly add 24 ml of diluted estradiol antibody under rapid stirring, stir for 10 min, add bovine serum albumin (BSA) to a final concentration of 1%, and stir for another 10 min. The initially prepared colloidal gold probe is Centrifuge at 4000r/mm for 20min; discard the precipitate, centrifuge the supernatant at 10000r/min for 60min; discard the supernatant and resuspend the pellet with 001mol/L phosphate buffer (PBS) containing 1% bovine serum albumin; After washing twice, the pellet was suspended in 001 mol/L PBS (pH 82, containing 1% BSA, 002% NaN3), and stored at 4 ° C until use. (4) Synthesis of antigen The small molecule substance is difficult to fix on the nitrocellulose membrane, and it can be well fixed only by attaching it to a macromolecular substance. In this experiment, a complete antigen was prepared by a mixed acid anhydride method. 43 mg of estradiol-6-oxime, 50 μl of tri-n-butylamine, 4 ml of dioxane, cooling to 10 ° C or less in an ice bath, and 15 μl of ethyl chloroformate [6]. The reaction was carried out at 4 to 10 ° C for 30 min to prepare an ovalbumin (OVA) solution (OVA 10 mg, 3 ml of water, 3 ml of dioxane, 1 mol/L of sodium hydroxide 03 ml). The prepared OVA solution was added to the reaction solution, and stirred in an ice bath at 4 ° C for 6 h. During the reaction, the pH was maintained with sodium hydroxide. After the reaction was completed, the reaction solution was added to a dialysis bag, and dialyzed for 2 d. The dialysate was adjusted to pH 45, refrigerator 4 ° C, placed for 4 days, a yellow precipitate appeared. The precipitate was collected by centrifugation, lyophilized, and stored in a refrigerator at 4 °C. The OVA was dissolved in the dialysate as a reference, and the estradiol-6è‚Ÿ-OVA was qualitatively examined by ultraviolet spectroscopy. The protein has been shown to bind to the estradiol derivative by UV measurement. (5) Preparation of colloidal gold immunochromatographic test strip The test strip consists of a glass fiber membrane, a nitrocellulose membrane, a sample paper, and an absorbent paper. The glass fiber membrane was cut into 6 mm thin strips, then immersed in PB solution containing 1% BSA, 1% Tween-20 for 30 min, dried at 37 ° C, and finally the colloidal gold probe was poured into the treated glass fiber membrane. , vacuum dry for use. The above antigen and rabbit anti-mouse IgG were sprayed into two lines on a nitrocellulose membrane by a spotting machine, which were respectively a detection line and a control line, and after vacuum drying, blocked with 1% BSA, 001 mol/L PBS (pH 90) for 2 h. It was washed with 001 mol/L PBS and dried under vacuum. A 30 mm absorbent paper, a 25 mm nitrocellulose membrane, a 6 mm glass fiber membrane, and a 15 mm sample paper were sequentially adhered to the PVC panel from the top, and cut into thin strips for use. (6) Test and interpretation samples: Ethanol solutions containing known concentrations of estradiol samples were divided into 3 groups, the first group was without estradiol, the second group was 02 μg/ml estradiol, and the third group was 04 μg/ Ml estradiol. Insert one end of the sample paper into the liquid to be tested, remove it after wetting, and place it horizontally for about 3 to 5 minutes to observe the result. For example, the test paper strip nitrocellulose membrane only has a purple-red band on the control line as positive; if two purple-red bands appear as negative; if the quality control line does not appear purple-red band, the test result is invalid regardless of whether the test line appears. Dried Purple Potato,Dried Purple Sweet Potato,Ad Purple Sweet Potato,Dehydrated Purple Sweet Potato Cube,Dehydrated Sweet Potato Jiangsu Tiankang Food Co., Ltd. , https://www.tiankangfood.com