CN104062441B - Method for measuring content of steroid metabolites in dung excrements of sheep - Google Patents

Abstract

The present invention relates to the technical field of scientific research and experimentation, in particular to a method for measuring the content of steroid metabolites in sheep feces excreta. A self-prepared reagent is used to measure the content of fecal steroid metabolites. The steps of determination and analysis are as follows: 1 ) reagent preparation; 2) microwell plate coating protein A; 3) storage solution preparation; 4) working solution preparation; 5) analysis steps; 6) absorbance measurement and calculation. The method of using self-prepared reagents to determine the content of steroid metabolites in sheep feces is mainly based on self-prepared reagents in the laboratory to save costs. And it can accurately measure the content of steroid metabolites. The invention provides a method for measuring the content of steroid metabolites in feces and excretions of sheep, which uses non-tissue damage sampling, avoids the influence of blood collection on the content of steroid hormones, and avoids damage to carcasses.

Description

Method for Determination of Steroid Metabolites in Sheep Fecal Excreta

technical field

The invention relates to the field of scientific research and experimentation, in particular to a method for measuring the content of steroid metabolites in sheep feces.

Background technique

At present, studies on the determination of steroid metabolites in fecal excreta mainly focus on wild animals and rare birds, and there are very few studies on domestic animals. However, the collection of feces and excreta of wild animals and rare birds requires blood collection or tissue damage sampling, which will cause damage to the carcass.

At present, the determination of steroid metabolites in feces is mainly used in kits, which are expensive and time-consuming.

Contents of the invention

In order to solve the shortcomings in the above technical problems, the present invention provides a method for measuring the content of steroid metabolites in sheep feces excrement. The method is mainly based on self-prepared reagents in the laboratory to save costs.

The technical scheme that solves the above-mentioned technical problem is:

The method for measuring the content of steroid metabolites in sheep feces excreta adopts a self-prepared reagent to measure the content of fecal steroid metabolites, and the steps of determination and analysis are as follows:

1) Preparation of reagents;

2) The microwell plate is coated with protein A;

3) Preparation of storage solution;

4) Preparation of working solution;

5) analysis steps;

6) Determination and calculation of absorbance.

Further, the preparation step of the described self-preparation reagent has the following nine aspects:

1) For the preparation of the first coating solution, 1.59 g of Na ₂ CO ₃ and 2.93 g of NaHCO ₃ were dissolved in double distilled water, and the pH was adjusted to 9.6;

2) Prepare 1mol/L of HCL;

3) Preparation of buffer solution: 2.42g Tris, 20mmol/L; 17.9g NaCl, 0.3mol/L; 1g bovine serum albumin; 1ml Tween 80; adjust the pH to 7.5;

4) Preparation of the second coating liquid, measure 3.146g of trishydroxyaminomethane; 23.3g of NaCl; 13g of BSA; 1.3g of sodium azide, dissolve in double distilled water, and prepare 1.3L, and adjust the pH to 7.5;

5) Preparation of cleaning solution, 0.5ml Tween 20, add 2.5L double distilled water;

6) Preparation of horseradish peroxidase substrate buffer solution, prepare 10mmol/l sodium acetate solution, take 1.36g of the solution and dissolve it in double distilled water, and make it into 1L, use about 8ml of 5% citric acid to adjust the pH Adjust to 5.0;

7) The preparation of the enzyme concentrated solution of streptavidin reaction, get above-mentioned step 3) prepared buffer solution 30ml and 0.001ml streptavidin-horseradish peroxidase conjugate on the magnetic stirrer well mixed;

8) The substrate solution preparation of horseradish peroxidase, get the substrate buffer solution of 30ml, 0.5ml3,3',5,5'-tetramethylbenzene (0.4%) and 0.1ml hydrogen peroxide (0.6% ) Gently mix on a magnetic stirrer;

9) Preparation of terminator, prepare 2mol/L sulfuric acid, take 900ml of double distilled water and 100ml of sulfuric acid (95-97%) and mix evenly.

Further, the preparation steps of protein A coated on the microwell plate are as follows: for a 96-well microplate, prepare 50 μg of protein A solution, dissolve it in the first coating solution of 25 ml (2 μg protein A/ml ), distribute the diluted protein A on the microplate, 0.25ml per well, incubate the microplate overnight at room temperature, discard the original solution, and refill the well with the second coating solution, 0.3ml per well , Cover the filled microwell plate with parafilm and a dust cover, and store at room temperature until use, which can be used after 3 hours.

Do not store microplates at room temperature for more than 4 weeks. (Dry the plate and freeze it at -20 degrees to keep the microplate longer)

Further, the preparation method of the storage solution is as follows: dilute 0.01ml storage solution (1mg steroid: 1ml methanol) with 20ml buffer solution, mix in an ultrasonic bath for 2 minutes, let stand for 3 hours, and use 0.05ml per bottle Dispensed into new vials, each containing 25,000 pg of the corresponding steroid.

All storage solutions were stored at -20°C until use. Waterproof color labels are affixed to the vials to indicate the different components: eg: Blue: Antibody (AB); Red: Biotinylated Steroid (BL); Green: Standard.

Further, the preparation method of the working solution is as follows: distribute 0.15ml of buffer solution in each bottle to some standard vials, vibrate and let stand for 20 minutes, dilute the solution 7 times with a ratio of 1:2.5, and in each step Both should be fully mixed (500pg/0.01ml-2pg/0.01ml).

This can be done with a Hamilton Microlab dispenser 1000 (0.09ml standard + 0.135ml buffer), or the entire standard vial (0.05ml) can be mixed with 0.15ml buffer and an additional 2.3ml buffer added (liquid must be transferred to a larger vial middle). The concentration of this result is 500pg/50μl buffer solution, and then further dilution (1:2.5; 1ml+1.5ml buffer solution), in some very sensitive assays, 9 dilutions are made, and the first two dilutions (80pg /0.01ml-0.3pg/0.01ml). Standards, antibodies, and biotin labeling solutions must be incubated for 20 minutes before use.

Antibodies and Biotinylated Steroids: For each individual EIA, see the specific label.

Further, the analysis steps include the following ten aspects:

1) Extract feces, and then pre-treat the extract;

Samples were frozen immediately after stool collection and stored at -20°C until analysis.

Weigh 0.5g of wet excrement plus 5ml of 80% methanol (pre-prepared, or mix with 4ml of 100% methanol and 1ml of water), in a multi-tubular vortex mixer for 30min (or in a manual vortex mixer for 1- 2min, then 15min in a centrifuge, 2500g);

2) Clean the microplate;

Before use, wash the coated microplate three times with washing solution, absorb the remaining liquid with absorbent paper, and dry the microplate. Be careful not to touch the bottom of the microplate.

3) Pipetting of standard solution and sample;

On the microplate, add 0.05ml of buffer solution to each blank well; add 50μl of pre-prepared standard solution to each standard solution well; add 0.01ml sample and 0.04ml buffer solution to each sample well, a total of 0.05ml per well; Each hole is 0.05ml, which can be distributed with Hamilton Mircolab dispenser1000.

4) Distribution of biotin-labeled steroids (BL);

Add 0.1 ml of biotinylated steroid to each well.

For all pipetting steps, use a multi-pipette (multi-tube pipette).

5) Distribution of antibody (AB);

Add 0.1ml of antibody solution to each well. (Note: Use buffer instead of AB for blank wells)

Cover the microplate with parafilm and dust cap and incubate overnight at 4°C with gentle shaking.

6) Wash the microwell plate after incubation;

Discard the liquid in the microwell plate after incubation, and wash the microwell plate 4 times with cold (4°C) washing solution.

7) streptavidin reaction;

Add 0.25ml of enzyme solution to each well, and place the sealed microplate on a shaker at 4°C for 45 minutes.

8) Wash the microwell plate again; the steps are the same as the above step 6);

9) color reaction;

Add 0.25 ml of substrate solution to each well, and incubate the sealed-covered microplate at 4°C for 45 minutes (with agitation).

10) terminate the reaction;

Add 0.05ml of terminator per well (blue to yellow).

Furthermore, the method for measuring and calculating the absorbance is as follows: use an automatic microwell plate connected to a computer, equipped with special calculation software, measure under the condition that the transmittance wavelength is 450nm, and calculate .

The above-mentioned scheme is adopted, and the method of measuring the content of steroid metabolites in sheep feces excreta with self-prepared reagents is mainly based on self-prepared reagents in the laboratory to save costs. And it can accurately measure the content of steroid metabolites. The invention provides a method for measuring the content of steroid metabolites in feces and excreta of sheep, which uses non-tissue damage sampling, avoids the influence of blood collection on the content of steroid hormones, and avoids damage to carcasses.

Description of drawings

Figure 1 is the input standard concentration value and absorbance filter value

Figure 2 is the regression curve

Figure 3 is the regression equation

Figure 4 is the input sample Y value

Figure 5 is the calculated sample concentration value

detailed description

The present invention will be described in further detail below in combination with specific embodiments.

1. Reagent preparation

1.1 The first coating solution: Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, dissolved in double distilled water, adjusted to pH 9.6;

1.21mol/LHCl;

1.3 Buffer: 2.42g Tris, 20mmol/L; 17.9g NaCl, 0.3mol/L; 1g bovine serum albumin; 1ml Tween 80; adjust the pH to 7.5;

1.4 The second coating solution: 3.146g tris; 23.3g NaCl; 13g BSA; 1.3g sodium azide, dissolved in double distilled water, and made into 1.3L, and the pH was adjusted to 7.5.

1.5 Cleaning solution: 0.5ml Tween 20, add 2.5L double distilled water.

1.6 Horseradish peroxidase substrate buffer solution: 1.36g of sodium acetate = 10mmol/l, dissolved in double distilled water, and made into 1L, adjusted to pH 5.0 with about 8ml of 5% citric acid.

1.7 Enzyme concentrated solution for streptavidin reaction: 30ml buffer + 0.001ml streptavidin-horseradish peroxidase conjugate (0.5U);

1.8 Substrate solution for horseradish peroxidase: 30ml substrate buffer + 0.5ml 3,3',5,5'-tetramethylbenzene (0.4%) + 0.1ml hydrogen peroxide (0.6%) before use Mix gently on a magnetic stirrer for several minutes. (This working solution must be ready-to-use)

1.9 Terminator: 2mol/L sulfuric acid, 900ml double distilled water + 100ml sulfuric acid (95-97%)

2. Microplate coated with Protein A

For a 96-well microplate, prepare 50 μg of protein A solution, dissolve it in 25 ml of coating solution (2 μg protein A/ml), distribute diluted protein A on the microplate, 0.25 ml per well, and place at room temperature Culture the microwell plate overnight, discard the original solution, and refill the well with the second coating solution, 0.3ml per well, cover the filled microwell plate with a parafilm and a dust cover, and store at room temperature until use. Can be used after 3 hours. Do not store microplates at room temperature for more than 4 weeks. (Dry the plate and freeze it at -20 degrees to keep the microplate longer)

3 Reagents (storage solution): All storage solutions are stored at -20 degrees until use. Waterproof color labels are affixed to the vials to indicate the different components: Example: blue: antibody (AB); red: biotinylated steroid (BL), green: standard; dilute 0.01ml of stock solution (1mg steroid) with 20ml buffer : 1ml methanol). Mix in an ultrasonic bath for 2 minutes, let stand for 3 hours, and dispense 0.05ml into new vials. Each vial contains 25,000 pg of the corresponding steroid.

4 working fluid

4.1 Standard solution

Distribute 0.15ml of buffer solution per bottle to some standard vials, shake and let stand for 20 minutes.

Dilute the solution 7 times at a ratio of 1:2.5, mixing thoroughly at each step (500pg/0.01ml-2pg/0.01ml). Nine dilutions were performed, and the first two dilutions (80pg/0.01ml-0.3pg/0.01ml) were discarded. Standards, antibodies, and biotin labeling solutions must be incubated for 20 minutes before use.

4.2 Antibodies and biotinylated steroids

For each individual EIA, go to the specific tab.

5 analysis steps

5.1 Extraction

Samples were frozen immediately after stool collection and stored at -20°C until analysis.

Weigh 0.5g of wet excrement plus 5ml of 80% methanol (pre-prepared, or mix with 4ml of 100% methanol and 1ml of water), in a multi-tubular vortex mixer for 30min (or in a manual vortex mixer for 1- 2min, then 15min in a centrifuge, 2500g);

5.2 Cleaning the microplate

Before use, wash the coated microplate three times with washing solution, absorb the remaining liquid with absorbent paper, and dry the microplate. Be careful not to touch the bottom of the microplate.

5.3 Standard solution and sample pipetting

On the microplate, add 0.05ml buffer to each blank well;

Add 50 μl of pre-prepared standard solution to each standard solution well;

Add 0.01ml sample + 0.04ml buffer solution to each sample well, a total of 0.05ml per well; each well is 0.05ml.

5.4 Distribution of biotinylated steroids (BL)

Add 0.1 ml of biotinylated steroid to each well.

For all pipetting steps, use a multi-pipette (multi-tube pipette).

5.5 Assignment of Antibody (AB)

Add 0.1ml of antibody solution to each well. (Note: Use buffer instead of AB for blank wells)

Cover the microplate with parafilm and dust cap and incubate overnight at 4°C with gentle shaking.

5.6 Washing the microplate after incubation

Discard the liquid in the microwell plate after incubation, and wash the microwell plate 4 times with cold (4°C) washing solution.

5.7 Streptavidin reaction

Add 0.25ml of enzyme solution to each well, and place the sealed microplate on a shaker at 4°C for 45 minutes.

5.8 The second cleaning is the same as 5.6

5.9 Color reaction

Add 0.25 ml of substrate solution to each well, and incubate the sealed-covered microplate at 4°C for 45 minutes (with agitation).

5.10 Termination reaction

Add 0.05ml of terminator per well (blue to yellow).

6 Absorbance measurement and calculation

Using an automatic microplate reader (connected to a computer, equipped with a commercially available ELISA Calc regression/fitting calculation program-v0.1 calculation software), the light transmittance is measured at 450nm and calculated.

The method for measuring and calculating the absorbance is as follows: put the terminated microplate into a microplate reader, select a wavelength of 450nm to measure the absorbance value; then use the ELISA Calc regression/fitting calculation program-v0.1 (Shanghai Provided by Biowo Biotechnology Co., Ltd.), calculate the regression equation through the concentration of the standard substance and the absorbance filter value, and then use the absorbance filter value (Y value) of the sample to calculate the concentration value (X value).

Specific examples are as follows:

Determination of estradiol concentration in feces.

1. Find the regression equation

(1) Input the concentration value (X value) and the absorbance filter value (Y value) of the standard substance, as shown in Figure 1, the results in Table 1 are obtained.

Table 1

Standard concentration (X value) Absorption filter value (Y value) 0pg 1.714 10pg 1.684 50pg 1.365 100pg 1.034 250pg 0.363 500pg 0.157 1000pg 0.063

Figure 1 Input standard concentration value and absorbance filter value

Click "Regression/Fitting" to get the regression curve and regression equation, as shown in Figures 2 and 3.

2. Find the concentration value of the sample

Table 2

Sample concentration (X value) Absorption filter value (Y value) 64.39 1.258 84.36 1.100 1.219 1.158 1.229 0.987 1.046

(1) Input the sample absorption filter value (Y value), as shown in Figure 4

Click "Calculate" to get the sample concentration value, as shown in Figure 5.

Figure 5 Calculated sample concentration value

(2) Calculate the concentration values of all samples sequentially as shown in Figures 4 and 5.

Claims (2)

1. measure the method for steroid metabolite content in sheep excrement, it is characterized in that, adopt a kind of self-prepared reagent to measure the content of feces steroid metabolite, step is successively: (1) Preparation of reagents; The preparation step of described self-preparation reagent has the following nine aspects: 1) For the preparation of the first coating solution, 1.59 g of Na ₂ CO ₃ and 2.93 g of NaHCO ₃ were dissolved in double distilled water, and the pH was adjusted to 9.6; 2) Prepare 1mol/L of HCl; 3) Preparation of buffer solution: 2.42g Tris, 20mmol/L; 17.9gNaCl, 0.3mol/L; 1g bovine serum albumin; 1ml Tween 80; adjust the pH to 7.5; 4) Preparation of the second coating solution, measure 3.146g tris; 23.3g NaCl; 13g BSA; Dissolve 1.3g of sodium azide in double distilled water to make 1.3L, and adjust the pH to 7.5; 5) Preparation of cleaning solution, 0.5ml Tween 20, add 2.5L double distilled water; 6) The preparation of horseradish peroxidase substrate buffer solution, the sodium acetate solution of preparation 10mmol/l, gets this solution 1.36g and dissolves with double distilled water, and is made into 1L, adjusts pH with 5% citric acid 8ml to 5.0; 7) For the preparation of streptavidin-horseradish peroxidase concentrated solution, 30ml of buffer solution prepared in the above step 3) and 0.001ml streptavidin-horseradish peroxidase conjugate Mix well on a magnetic stirrer; 8) The substrate solution of horseradish peroxidase is prepared by taking 30ml of horseradish peroxidase substrate buffer, 0.5ml of 3,3',5,5'-tetramethylbenzene 0.4% and 0.1ml of 0.6 % Hydrogen peroxide was gently mixed on a magnetic stirrer; 9) The preparation of terminator, prepare 2mol/L sulfuric acid, take 900ml of double distilled water and 100ml of 95-97% sulfuric acid and mix evenly; (2) The microwell plate is coated with protein A; The preparation steps of the protein A coated on the microplate are as follows: for a 96-well microplate, prepare 50 μg of protein A solution, dissolve it in 25 ml of the first coating solution, 2 μg protein A/ml, distribute the diluted Protein A on the microwell plate, 0.25ml per well, culture the microwell plate overnight at room temperature, discard the original solution, and refill the well with the second coating solution, 0.3ml per well, cover with parafilm and Cover the filled microplate with a dust cover and store at room temperature until use; (3) Preparation of storage solution; the preparation method of the storage solution is: dilute 0.01ml of 1mg steroid with 20ml of buffer: 1ml of methanol storage solution, mix in an ultrasonic bath for 2 minutes, let stand for 3 hours, press each Bottle 0.05ml is divided into new vials, each vial contains the corresponding steroid 25000pg; (4) Preparation of standard solution; the preparation method of said standard solution is: distribute 0.15ml damping solution in every bottle in some standard vials, shake and stand for 20 minutes, dilute this solution 7 times with the ratio of 1: 2.5, Mix the buffer thoroughly at each step; (5) Analysis steps; The analysis steps include the following: 1) Extract feces, and then pre-treat the extract; 2) Clean the microplate; 3) Pipetting of standard solution and sample; 4) Distribution of biotinylated steroids; 5) Distribution of antibody Ab; 6) Wash the microwell plate after incubation; 7) streptavidin-horseradish peroxidase conjugate reaction; 8) Wash the microplate again; 9) color reaction; 10) Terminate the reaction; after terminating the reaction, obtain: the color of each hole solution is a yellow microplate with different degrees, and then put it into the microplate reader to set the wavelength and the position of the blank hole, and measure the absorbance value of each hole of the microplate ; (6) Absorbance measurement and calculation; The method for measuring and calculating the absorbance is as follows: connect an automatic microplate reader to a computer, equip with commercially available ELISA Calc regression/fitting calculation program-v0.1 calculation software, measure the absorbance at 450nm, and calculate . 2. the method for the content of steroid metabolites in mensuration sheep excrement according to claim 1 is characterized in that, the absorbance measurement described in step (6) and the method for calculating are: the microporous plate of termination reaction is put into In the microplate reader, select a wavelength of 450nm to measure the absorbance value; then use the ELISA Calc regression/fitting calculation program-v0.1, provided by Shanghai Biowo Biotechnology Co., Ltd. to calculate the regression equation through the standard concentration and absorbance value, and then use the absorbance of the sample The value Y value calculates the concentration value X value.