Estradiol Detection Laboratory
Estradiol Detection Laboratory
Estradiol (17β-Estradiol) is an important component of natural estrogen, and the detection of estradiol can be used for (1) the determination of hormone additives in food safety (2) the addition of illegal hormones to aquaculture feeds (3) the rapid determination of clinical samples.
Operation method
immunocolloid gold paper method
Principle
Using colloidal gold particles labeled with monoclonal antibodies to estradiol combined with corresponding estradiol conjugates immobilized on a nitrocellulose membrane, the detection line with the immobilized estradiol conjugates shows a distinct color.
Materials and Instruments
Estradiol Antibodies Move I. Reagents and Instruments Caveat 1. If the operator's skin or clothing is contaminated (stained) with blood or waste liquid, rinse and disinfect it immediately. If the eyes are splashed with blood or waste liquid they should be rinsed immediately with plenty of water and necessary medical treatment should be carried out. 2. All blood specimens should be capped during centrifugation, and after centrifugation, care should be taken to prevent contamination of the environment by aerosols (mist) when opening the tubes. 3. all tested blood specimens and related wastes are potentially dangerous and biologically contaminating to you. Dispose of all discarded specimens and waste in the same way as for blood specimens. Common Problems This method has the advantages of (1) high sensitivity, (2) high specificity, (3) simplicity and rapidity, (4) low cost, and (5) easy to interpret the results, and it is suitable for the preliminary screening of samples. For more product details, please visit Aladdin Scientific website.
Chloroauric Acid Bovine Serum Albumin Ovalbumin Rabbit Anti-mouse Polyantibody Estradiol
Nitrocellulose membrane Glass fiber membrane Spectrophotometer Low-temperature high-speed centrifuge Spotting machine
Chloroauric acid; bovine serum albumin, ovalbumin, rabbit anti-mouse polyantibody; estradiol standard, estradiol monoclonal antibody, 17β-Estradiol-6-one6-(o-carboxymethvyoxime). Nitrate fiber membrane, glass fiber membrane; Beckman Du530 spectrophotometer; low-temperature high-speed centrifuge; film dispenser.II. Methods
1. Preparation of colloidal gold
Dissolve the chloroauric acid with triple distilled water to make the final concentration of 01g/L. After the chloroauric acid solution was boiled, add 25 ml of 1% trisodium citrate for every 100 ml, and then stir rapidly under the boiling water bath until the color of the chloroauric acid solution stabilized, and then continue the boiling water bath for 10 min, and then cooled down to room temperature, and then examined the particles uniformity and particle size by transmission electron microscope and spectrophotometer. Finally, it was stored in a refrigerator at 4℃ for spare parts.2. Determination of the optimal amount of labeled protein
The colloidal gold solution was adjusted to pH 8.2 with 0.2 mol/L K2CO3, and then 9 test tubes were taken and 10 ml of colloidal gold solution was added respectively. After the estradiol antibody was diluted step by step, an equal volume of dilution was added into the above test tubes sequentially and mixed well, and a control tube without antibody was set up. After 10 min, add 0.1 ml of 10% NaCl into each tube, mix well and leave for 2 h. Observe the changes of colloidal gold solution in each tube, the color of the solution without protein and with insufficient amount of added protein changed from red to blue, while the solution with the amount of added protein reached or exceeded remained unchanged. The amount of labeled protein was 20% of the amount of protein added to the lowest stabilized colloidal gold solution that did not change color.3. Colloidal gold probe labeling
The antibody was dialyzed overnight in 0005 mol NaCl solution, centrifuged to remove the protein precipitate, and adjusted to 05 mg/ml. 100 ml of colloidal gold was taken, and the colloidal gold solution was adjusted to pH 9.0 with 0.2 mol/L K2CO3, and 24 ml of diluted estradiol antibody was added slowly under rapid magnetic stirring, and stirring was continued for 10 min, and bovine serum albumin (BSA) was added so that the final concentration was 1%, and stirring was continued. The preliminary colloidal gold probe was centrifuged at 4 000 r/mm for 20 min; the precipitate was discarded, and the supernatant was centrifuged at 10 000 r/min for 60 min; the supernatant was discarded, and the precipitate was resuspended in 0.1 mol/L phosphate buffer solution (PBS) (containing 1% bovine serum albumin); the same centrifugation was washed twice, and the precipitate was washed in 0.1 mol/L PBS for 2 times. mol/L PBS (pH 82, containing 1% BSA, 002% NaN3 ) and stored at 4°C for spare parts.4. Antigen synthesis
Small molecules are difficult to be fixed on the nitrocellulose membrane, only make it attached to the large molecules can be better fixed. In this experiment, complete antigen was prepared by mixed anhydride method. Estradiol-6-oxime 43 mg was added to tri-n-butylamine 5 ul, dioxane 4 ml, cooled to below 10°C in an ice bath, and ethyl chloroformate 15 ul. The reaction was carried out for 30 min at 4-10°C. Ovalbumin (OVA) solution (OVA 10 mg, 3 ml of water, 3 ml of dioxane, and 1 mol/L sodium hydroxide 03 ml) was prepared. 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, sodium hydroxide was used to maintain the pH at 8. After the reaction was completed, the reaction solution was added to a dialysis bag, and dialyzed for 2 d. The dialysis solution was adjusted to pH 4.5, refrigerated at 4°C, and placed in the refrigerator for 4 days, and a yellow precipitate appeared. The precipitate was collected by centrifugation, freeze-dried and stored in the refrigerator at 4 °C. OVA was dissolved in the dialysate as a reference, and estradiol-6oxime-OVA was qualitatively examined by UV spectroscopy. The protein was proved to be bound to the estradiol derivative by UV determination.5. Colloidal gold immunochromatographic test strip preparation
The test strips consisted of 4 parts: glass fiber membrane, nitrate fiber membrane, spiked paper and absorbent paper. The glass fiber membrane was cut into 6 mm thin strips, and then put into the PB solution containing 1% BSA, 1% Tween-20 immersed in 30 min, drying at 37 ℃, and finally the colloidal gold probe perfusion has been treated on the glass fiber membrane, vacuum drying standby. The above antigen and rabbit anti-mouse IgG were sprayed into 2 lines on the nitrate fiber membrane with a dotting machine, which were the detection line and the control line, respectively, and after vacuum drying, the membrane was closed with 1% BSA, 0.1 mol/L PBS (pH 9.0) for 2 h, washed with 0.1 mol/L PBS, and then vacuum dried. The 30 mm absorbent paper, 25 mm nitrate fiber membrane, 6 mm glass fiber membrane, and 15 mm spiked paper were glued to the PVC board sequentially from the top and cut into thin strips for spare use.6. Detection and interpretation of samples
The ethanol solution containing samples with known concentrations of estradiol was divided into 3 groups, group 1 without estradiol, group 2 with 0.2 ug/ml estradiol, and group 3 with 0.4 ug/ml estradiol. Insert one end of the spiked paper into the liquid to be tested, remove it after wetting, place it horizontally for about 3-5 min and observe the results. If there is only one purple-red band on the control line on the nitrocellulose membrane of the test strip, it is positive; if there are two purple-red bands, it is negative; if there is no purple-red band on the quality control line, the test result is invalid regardless of the presence or absence of the test line.