阅读说明：本技术 一种标准品稀释液的简易制备方法 (Simple preparation method of standard product diluent ) 是由 黄振宇 于 2020-10-23 设计创作，主要内容包括：本发明公开了一种标准品稀释液的简易制备方法,包括以下步骤：步骤一：在成分明确的缓冲液中,加入不同浓度的生化试剂,浓度范围为0％-10％,浓度梯度为0.5％；步骤二：在各个浓度的标准品稀释液和若干待测样本中,加入等量高浓度的目标蛋白,加入目标蛋白的体积不超过整个体系的10％,并使加入目标蛋白的终浓度位于标曲范围的中段；步骤三：对加入目标蛋白后的标准品稀释液、加入目标蛋白后的待测样本以及不加目标蛋白的待测样本按常规ELISA实验方案操作；步骤四：绘制OD曲线：横坐标为不同浓度的标准品稀释液,纵坐标为OD值；本发明的标准品稀释液成分明确、制备方法相对简易,该配方的标准品稀释液不易长菌,具有突出性的进步。(The invention discloses a simple preparation method of a standard product diluent, which comprises the following steps: the method comprises the following steps: adding biochemical reagents with different concentrations into a buffer solution with definite components, wherein the concentration range is 0-10%, and the concentration gradient is 0.5%; step two: adding target protein with equal high concentration into standard product diluent and a plurality of samples to be detected, wherein the volume of the added target protein is not more than 10% of the whole system, and the final concentration of the added target protein is positioned in the middle section of the standard curve range; step three: performing conventional ELISA experimental scheme operation on the standard substance diluent added with the target protein, the sample to be detected added with the target protein and the sample to be detected without the target protein; step four: drawing an OD curve: the abscissa is standard dilution liquid with different concentrations, and the ordinate is OD value; the standard product diluent has definite components and relatively simple preparation method, and the standard product diluent of the formula is not easy to grow bacteria and has outstanding progress.)

1. A simple preparation method of a standard product diluent is characterized by comprising the following steps:

the method comprises the following steps: adding biochemical reagents with different concentrations into a buffer solution with definite components, wherein the concentration range is 0-10%, and the concentration gradient is 0.5%;

step two: adding target protein with equal high concentration into standard product diluent and a plurality of samples to be detected, wherein the volume of the added target protein is not more than 10% of the whole system, and the final concentration of the added target protein is positioned in the middle section of the standard curve range;

step three: performing conventional ELISA experimental scheme operation on the standard substance diluent added with the target protein, the sample to be detected added with the target protein and the sample to be detected without the target protein;

step four: drawing an OD curve: the abscissa is standard dilution liquid with different concentrations, and the ordinate is OD value;

step five: determining standard substance diluent intersecting with the average value of the OD value of the sample to be detected, namely determining that the target protein has a similar effect of combining with the antibody in a standard substance diluent system represented by the intersection point and the sample to be detected;

step six: selecting the standard substance diluent screened in the step and the standard substance diluents with two adjacent concentrations as candidate standard substance diluents, taking a plurality of normal samples, selecting high, medium and low 3 concentrations in the range of the standard curve to perform a standard addition recovery experiment, confirming whether the recovery rate has deviation, and continuing to adjust the concentration until the recovery rate meets the standard.

2. The method for preparing a dilution of a standard substance according to claim 1, wherein in the second step, the samples to be tested are selected from the group consisting of serum of healthy human, mouse and rat.

3. The method of claim 1, wherein the recovery rate is in the range of 80-120%.

4. The method of claim 1, wherein the average value of the OD values of the samples to be measured is obtained by subtracting the OD value of the sample to be measured without the target protein from the OD value of the sample to be measured after the target protein is added.

5. The method of claim 1, wherein the 3 concentrations in the sixth step are final concentrations after the target protein is added, and correspond to the concentrations at 2 nd, 4 th and 6 th points in the standard curve range.

Technical Field

The invention relates to the technical field of accurate quantification of soluble target protein, in particular to a simple preparation method of a standard product diluent.

Background

ELISA (enzyme linked immunosorbent assay) is a classical immunological experiment and has the characteristics of high sensitivity, strong specificity and good repeatability.

Wherein the performance parameter for evaluating the ELISA accuracy is the recovery rate, namely the ratio of a calculated value of the target protein to a theoretical value shows the difference between the calculated value and a real value, and the recovery rate of an excellent kit is 80-120%; theoretically, the standard dilution should be identical to the matrix of the test sample except that it does not contain the target protein, but it is actually difficult to realize the method and it is more difficult to mass-produce the method; the substitute method is to obtain equivalent standard product diluent, the current common component is animal serum, and the serum sample system can be better simulated; however, animal serum has many disadvantages, such as large difference between batches of serum due to individual difference of animals, easy blockage of filter membrane and difficult filtration when serum concentration is high, easy growth of bacteria due to abundant protein content in serum, etc. in addition, because serum composition is complex, there are many unknown proteins, when problems occur, it is difficult to judge influencing factors, which brings great trouble to production research.

In summary, there is a need for a simple method for preparing a standard dilution solution that solves the problems of the prior art.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a simple preparation method of a standard product diluent, aiming at solving the problems.

In order to achieve the purpose, the invention provides the following technical scheme: a simple preparation method of a standard product diluent comprises the following steps:

the method comprises the following steps: adding biochemical reagents with different concentrations into a buffer solution with definite components, wherein the concentration range is 0-10%, and the concentration gradient is 0.5%;

step two: adding target protein with equal high concentration into standard product diluent and a plurality of samples to be detected, wherein the volume of the added target protein is not more than 10% of the whole system, and the final concentration of the added target protein is positioned in the middle section of the standard curve range;

step three: performing conventional ELISA experimental scheme operation on the standard substance diluent added with the target protein, the sample to be detected added with the target protein and the sample to be detected without the target protein;

step four: drawing an OD curve: the abscissa is standard dilution liquid with different concentrations, and the ordinate is OD value;

step five: determining standard substance diluent intersecting with the average value of the OD value of the sample to be detected, namely determining that the target protein has a similar effect of combining with the antibody in a standard substance diluent system represented by the intersection point and the sample to be detected;

step six: selecting the standard substance diluent screened in the step and the standard substance diluents with two adjacent concentrations as candidate standard substance diluents, taking a plurality of normal samples, selecting high, medium and low 3 concentrations in the range of the standard curve to perform a standard addition recovery experiment, confirming whether the recovery rate has deviation, and continuing to adjust the concentration until the recovery rate meets the standard.

Furthermore, in the second step, one of the serum of a healthy person, a mouse or a rat is adopted as the samples to be detected.

Further, the confirmed recovery rate ranges from 80 to 120%.

Further, the average value of the OD values of the samples to be detected is obtained by subtracting the OD value of the sample to be detected without the target protein from the OD value of the sample to be detected after the target protein is added.

Further, 3 concentrations in the sixth step are final concentrations after the target protein is added, and correspond to the concentrations at the 2 nd, 4 th and 6 th points in the range of the standard curve.

The invention has the beneficial effects that: the standard product diluent has definite components and a relatively simple preparation method, the biochemical reagent with high purity and definite components is used for replacing serum to ensure the stability among batches, the biochemical reagent has the effect equivalent to that of the serum at lower concentration, in addition, the standard product diluent of the formula is convenient to prepare, remove impurities and carry out sterile filtration, and the standard product diluent is difficult to grow bacteria due to low protein content, so that the standard product diluent has prominent progress.

Drawings

FIG. 1 is a graph showing OD curves of dilutions of standard sodium iodide at different concentrations.

FIG. 2 is a graph showing the OD curves of 2% gelatin containing sodium pyrophosphate in different concentrations as standard dilutions.

FIG. 3 is a graph of OD curves for dilutions of different concentrations of BSA as standard.

Detailed Description

As shown in fig. 1, 2 and 3, a simple preparation method of a standard product diluent comprises the following steps:

the method comprises the following steps: adding biochemical reagents with different concentrations into buffer solution (such as PBS, PBST and Tris) with definite components, wherein the concentration range is 0-10%, and the concentration gradient is 0.5%; or selecting buffer containing high purity protein (such as BSA, gelatin) with concentration range of 0% -40% and concentration gradient of 5% (such as BSA), or with concentration range of 0% -10% and concentration gradient of 0.5% (such as gelatin), adding biochemical reagents with different concentrations with concentration range of 0% -10% and concentration gradient of 0.5%.

Step two: adding target protein with equal high concentration into standard product diluent and a plurality of samples to be detected, wherein the volume of the added target protein is not more than 10% of the whole system, and the final concentration of the added target protein is positioned in the middle section of the standard curve range;

step three: performing conventional ELISA experimental scheme operation on the standard substance diluent added with the target protein, the sample to be detected added with the target protein and the sample to be detected without the target protein;

step four: drawing an OD curve: the abscissa is standard dilution liquid with different concentrations, and the ordinate is OD value;

step five: determining standard substance diluent intersecting with the average value of the OD value of the sample to be detected, namely determining that the target protein has a similar effect of combining with the antibody in a standard substance diluent system represented by the intersection point and the sample to be detected;

step six: selecting the standard substance diluent screened in the step and the standard substance diluents with two adjacent concentrations as candidate standard substance diluents, taking a plurality of normal samples, selecting high, medium and low 3 concentrations in the range of the standard curve to perform a standard addition recovery experiment, confirming whether the recovery rate has deviation, and continuing to adjust the concentration until the recovery rate meets the standard.

Furthermore, in the second step, one of the serum of healthy human, mouse or rat is used as the samples to be detected.

Further, the recovery rate was confirmed to be in the range of 80 to 120%.

Further, the average value of the OD values of the samples to be detected is the OD value of the sample to be detected after the target protein is added minus the OD value of the sample to be detected without the target protein.

Further, 3 concentrations in step six are the final concentrations after addition of the target protein, corresponding to the concentrations at points 2, 4 and 6 in the range of the standard curve.

Example 1:

the screening of the standard dilution of the human IL-6ELISA kit comprises the following steps:

the method comprises the following steps: adding biochemical reagent (sodium iodide) with different concentration gradients into 1 XPBST, wherein the concentration gradient is 0.5%, and the concentration fractions are 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%, respectively;

step two: taking 120 mu l of each standard product diluent, and adding 5 mu l of recombinant human IL-6 protein with the concentration of 312.5pg/ml to make the final concentration of 12.5 pg/ml; taking 57.5 mu l of each of 8 normal human serums, adding 5 mu l of recombinant human IL-6 protein with the concentration of 312.5pg/ml to make the final concentration of 25 pg/ml;

step three: diluting the normal human serum added with the target protein and the normal human serum not added with the target protein by 2 times by using the same volume of detection buffer solution; adding 100 mul of standard dilution added with a standard and a diluted sample to be detected into each hole of a pre-coated IL-6 enzyme label plate; adding 50 mul of biotin-labeled anti-human IL-6 antibody into each hole, sealing the membrane, placing the membrane in a microplate oscillator, and oscillating and incubating for 2 hours at room temperature at 300 revolutions per minute; then washing 6 times with washing liquor, adding 100 mu l of Streptavdin-HRP into each hole, incubating for 45 minutes, washing 6 times, adding 100 mu l of TMB into each hole, developing for 10 minutes, and adding stop solution to stop the reaction; performing dual-wavelength detection in an enzyme-labeling instrument, wherein the dominant wavelength is 450nm, and the reference wavelength is 570nm or 630 nm;

step four: drawing an OD curve according to the reading result;

step five: according to the OD curve chart drawn in the fourth step, the average OD value of 8 normal human serum samples added with the standard substance is intersected with the buffer solution containing 1% of sodium iodide;

step six: the recovery rates of the normal human serum samples were adjusted to 3 concentrations using the buffer solutions containing 0.5%, 1% and 1.5% sodium iodide as the candidate standard dilutions, and the results showed that the recovery rates ranged from 93 to 110% and the average recovery rate was 102% using the buffer solution containing 1% sodium iodide as the standard dilution.

Furthermore, the IL-6 content in the serum of normal human is usually below 7pg/ml or is not detectable, and the data of the sample with higher content should be removed.

Example 2:

the screening of the standard dilution of the mouse TNF-alpha ELISA kit comprises the following steps:

the method comprises the following steps: adding biochemical reagents (sodium pyrophosphate) with different concentration gradients into 2% gelatin, wherein the concentration gradient is 0.5%, and the concentration fractions are 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%, respectively;

step two: mu.l of each standard dilution was taken and 0.75. mu.l of 40,000pg/ml recombinant mouse TNF-. alpha.protein was added to give a final concentration of 250 pg/ml. Taking 15 mu l of each of 8 normal mouse sera, adding 1 mu l of recombinant mouse TNF-alpha protein with the concentration of 40,000pg/ml to make the final concentration of 2500 pg/ml;

step three: the serum of normal mice to which the target protein was added and the serum of normal mice to which the target protein was not added were diluted 10-fold with the detection buffer. Adding 100 mul of standard dilution added with a standard and a diluted sample to be detected into each hole of a pre-coated mouse TNF-alpha enzyme label plate; adding 50 mul of biotin-labeled anti-mouse TNF-alpha antibody into each hole, sealing the membrane, placing the membrane in a microplate oscillator, and oscillating and incubating for 2 hours at room temperature at 300 revolutions per minute; then washing 6 times with washing liquor, adding 100 mu l of Streptavdin-HRP into each hole, incubating for 45 minutes, washing 6 times, adding 100 mu l of TMB into each hole, developing for 10 minutes, and adding stop solution to stop the reaction; performing dual-wavelength detection in an enzyme-labeling instrument, wherein the dominant wavelength is 450nm, and the reference wavelength is 570nm or 630 nm;

step four: drawing an OD curve according to the reading result;

step five: according to the OD curve chart drawn in the fourth step, the average OD value of 8 normal mouse serum samples added with the standard substance is intersected with the buffer containing 1% sodium pyrophosphate;

step six: the buffer solution containing 0.5%, 1% and 1.5% sodium pyrophosphate is used as candidate standard dilution, 10 normal mouse serum samples are taken for standard addition recovery of 3 concentrations, and the result shows that under the condition that the buffer solution containing 1% sodium pyrophosphate is used as standard dilution, the recovery rate is in the range of 88-107%, and the average recovery rate is 99%.

Furthermore, in the serum of normal mice, the content of TNF-alpha is usually low or undetectable, and the data of samples with high content should be eliminated.

Example 3:

the screening of the dilution of the human hypersensitive IFN-gamma ELISA kit standard substance comprises the following steps:

the method comprises the following steps: adding Bovine Serum Albumin (BSA) with different concentration gradients into 1 XPBST, wherein the concentration gradient is 5%, and the concentration fractions are 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40%, respectively;

step two: mu.l of each standard dilution was taken, and 0.6. mu.l of recombinant human IFN-. gamma.protein was added at a concentration of 2,000pg/ml to give a final concentration of 10 pg/ml. Taking 25 mu l of each 8 parts of healthy human serum, and adding 0.625 mu l of recombinant human IFN-gamma protein with the concentration of 2,000pg/ml to make the final concentration of 50 pg/ml;

step three: the serum of the healthy human to which the target protein was added and the serum of the healthy human to which the target protein was not added were diluted 5-fold with the detection buffer. Adding 100 mul of standard dilution and diluted sample to be tested into each hole in a pre-coated human high-sensitivity IFN-gamma enzyme label plate, sealing the membrane, placing the plate in a microplate oscillator, and oscillating and incubating the plate for 30 minutes at the room temperature at 300 r/min; washing with washing solution for 6 times, adding 100 μ l biotin-labeled anti-human IFN- γ antibody into each well, sealing, placing in a microplate oscillator, and oscillating and incubating at room temperature at 300 rpm for 30 min; washing with a washing solution for 6 times, adding 100 μ l of Streptavdin-HRP to each well, and incubating for 15 minutes; after washing, adding 100 mu l of signal enhancer into each hole, incubating for 15 minutes, washing for 6 times by using washing liquor, adding 100 mu l of Streptavdin-HRP into each hole, incubating for 15 minutes, adding 100 mu l of TMB into each hole after washing, developing for 10 minutes, and adding stop solution to stop reaction; performing dual-wavelength detection in an enzyme-labeling instrument, wherein the dominant wavelength is 450nm, and the reference wavelength is 570nm or 630 nm;

step four: drawing an OD curve according to the reading result;

step five: according to the OD curve chart drawn in the fourth step, the average OD value of 8 healthy human serum samples added with the standard substance is intersected with the buffer solution containing 30% BSA;

step six: the recovery rates of the spiked samples were 3 concentrations for 10 samples of healthy human serum using 25%, 30% and 35% BSA in the buffer as the candidate standard dilutions, and the results showed that the recovery rates ranged from 85 to 94% and the average recovery rate was 89% using 30% BSA in the buffer as the standard dilution.

Furthermore, the serum of healthy people has low or undetectable IFN-gamma content, and the data of samples with high content should be eliminated.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.