CN115343238A - Kit and method for determining content of lactic acid in biological sample - Google Patents

Abstract

The invention discloses a kit and a method for measuring the content of lactic acid in a biological sample, wherein the kit comprises a solvent consisting of a trihydroxymethyl aminomethane-hydrochloric acid solution, a lactic acid standard solution, a reagent I consisting of nicotinamide adenine dinucleotide, nitrotetrazolium blue and phenazine methyl sulfate, a reagent II consisting of lactic dehydrogenase and a terminator consisting of concentrated hydrochloric acid. The principle of the method is that lactic dehydrogenase and color developing agent are utilized to carry out electron transfer, the product presents purple red under the alkaline condition, an enzyme labeling instrument is utilized to measure the absorbance value, and the concentration of lactic acid is calculated. The method has the advantages of simple and easy sample pretreatment process, few reagent types, small sample consumption and cost saving. The method can be used for measuring a large number of samples, has good repeatability, high accuracy and good stability, and is suitable for being applied in a conventional laboratory.

Description

Kit and method for determining content of lactic acid in biological sample

Technical Field

The invention belongs to the technical field of lactic acid detection, and particularly relates to a kit and a method for determining the content of lactic acid in a biological sample.

Background

Lactic acid is closely related to sugar, lipid, protein metabolism and intracellular energy metabolism. Lactic acid is also an important product of glycolytic pathway, and can be converted into glucose through gluconeogenesis to supply body energy. It is thus seen that lactic acid is an important metabolite of the human body. The blood lactic acid can be used as a judgment index of the oxygen-containing condition of tissues and also can be used as an important index for estimating the shock prognosis. In addition, lactic acid is also associated with various diseases (metabolic diseases, tumors, liver diseases, etc.). Therefore, the determination and analysis of the lactic acid content in the biological sample have important significance for monitoring the occurrence and development of diseases.

There are three main types of methods for measuring lactic acid: chemical methods, gas chromatography, and enzymatic colorimetry. The reagent used in the chemical method is simpler, but the defects are that the operation is complicated, the time consumption is longer, the stability is poor, and the interference is easy to happen; the gas chromatography instrument is expensive, and the pretreatment of the biological sample is complex and tedious, so that the gas chromatography instrument is difficult to be widely applied in clinical laboratories. The enzyme colorimetric method has the advantages of good specificity, strong anti-interference capability, simple and convenient operation, less time consumption, economy, less sample consumption and the like, and is convenient for practical application. The enzymatic colorimetric method mainly comprises a lactic acid dehydrogenase method and a lactic acid oxidase method. At present, the enzyme colorimetric method has certain disadvantages. For example, the reaction reagent involved in the lactate dehydrogenase method is unstable, while the lactate oxidase method involves various enzymes to participate in the reaction, and the reaction reagent is more, the steps are complicated, and the method is not favorable for the determination of large clinical sample amount. Based on the method, the method for detecting the content of the lactic acid in the biological samples (blood, tissues and cells) by the simple, quick and stable enzymatic colorimetric method is invented. The reaction principle is as follows:

l-lactic acid + nicotinamide adenine dinucleotide

Pyruvic acid + reducing coenzyme I;

reductive coenzyme I + nitrotetrazolium blue

Nicotinamide adenine dinucleotide reduced nitrotetrazolium blue (purple).

The patent document with the publication number of CN110426357A discloses a high-sensitivity lactic acid detection reagent, the patent document with the publication number of CN110343740A discloses a lactic acid kit and a preparation method and application thereof, the patent document with the publication number of CN109957603A discloses a high-sensitivity lactic acid detection reagent, the three methods all adopt a multi-step enzyme reaction method, the reaction steps are complicated, and the reaction reagent contains some toxic substances, such as sodium azide, which is not beneficial to environmental protection.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the kit and the method for measuring the content of the lactic acid in the biological sample, and the kit and the method have the characteristics of simple, convenient, quick and effective operation, good repeatability, high accuracy and the like.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a kit for measuring the content of lactic acid in a biological sample comprises a first reagent and a second reagent,

a first reagent: is prepared from nicotinamide adenine dinucleotide, nitrotetrazolium blue and phenazine methyl sulfate, and is stored in powder of 1 bottle at-20 ℃;

and a second reagent: it is composed of lactate dehydrogenase, powder is multiplied by 1 bottle, and is preserved at-20 ℃.

Furthermore, the mass of nicotinamide adenine dinucleotide contained in the first reagent is 23.7 mg, the mass of nitrotetrazolium blue is 10.4 mg, and the mass of phenazine methosulfate is 3.56 mg.

Further, the mass of the lactate dehydrogenase contained in the second reagent is 15 mg.

Further, the solvent is composed of a tris-hydroxymethyl aminomethane-hydrochloric acid solution, the liquid is 100mL multiplied by 1 bottle, and the solution is stored at 4 ℃.

Further, the tris-hcl solution has a pH of 8.8.

Further, the lactic acid sample solution is also included, the concentration of the lactic acid sample solution is 5.56 mmol/L, and the lactic acid sample solution is stored at 4 ℃.

Further, the liquid comprises a terminating agent, wherein the terminating agent consists of hydrochloric acid, and the liquid is stored in a 50 mL X1 bottle at room temperature.

Further, the concentration of the hydrochloric acid is 1.0 mol/L.

Further, the kit also comprises a 96-pore plate, wherein 8 marked pores are used as main pores and are used for respectively adding lactic acid standard solutions with different concentrations; marking other holes as auxiliary holes, and respectively adding the solution to be detected and the other reagents and solvents.

The invention also provides a method for measuring the content of lactic acid in a biological sample, which comprises the following steps:

(1) Mixing a biological sample to be detected with a first reagent mainly comprising nicotinamide adenine dinucleotide, nitroblue tetrazolium and phenazine methyl sulfate and a second reagent comprising lactate dehydrogenase to react as follows:

l-lactic acid + nicotinamide adenine dinucleotide

Pyruvic acid + reducing coenzyme I

Reductive coenzyme I + nitrotetrazolium blue

Nicotinamide adenine dinucleotide + purple reduced nitro blue tetrazolium

(2) And detecting the absorbance of the final reactant at 550 nm, and calculating the content of the lactic acid in the sample.

Furthermore, the volume of the biological sample to be detected is 1-5 mu L, the reaction temperature is 37 ℃, and the reaction time is 15min.

Further, the reagent is dissolved by a solvent and is prepared as soon as used; the preparation process of the reagent I comprises the following steps: nicotinamide adenine dinucleotide at 23.7. Sup. 23.7 mg, nitrotetrazolium blue at 10.4. Sup. 10.4 mg, phenazine methosulfate at 3.56. Sup. 3.56 mg were weighed and dissolved in solvent 20 mL.

Further, the reagent is dissolved by a dual-purpose solvent and is prepared as soon as used; the preparation process of the reagent II comprises the following steps: the lactate dehydrogenase 15 mg was weighed out accurately and dissolved in 15 mL solvent.

Further, the solvent adopts a trihydroxymethyl aminomethane-hydrochloric acid solution with the pH value of 8.8, and the preparation process comprises the following steps: 1.213 g trihydroxymethylaminomethane is weighed, dissolved with double distilled water, and the pH is adjusted to 8.8 with concentrated hydrochloric acid, and finally the volume is fixed to 100mL with double distilled water, and the solution is stored in a refrigerator at 4 ℃ for 6 months.

Further, a lactic acid standard solution with the concentration of 5.56 mmol/L is added in the reaction process, and the preparation process comprises the following steps: accurately weighing 5.0 mg lactic acid, dissolving with solvent and making the volume to 10 mL, preparing into mother liquor with concentration of 5.56 mmol/L, storing in a tight-stoppered brown bottle, placing in a refrigerator at 4 deg.C, and storing for 6 months.

Furthermore, the reaction is stopped by adding a terminating agent in the reaction process, the terminating agent adopts hydrochloric acid with the concentration of 1.0 mol/L, and the preparation process comprises the following steps: taking 4.2 mL concentrated hydrochloric acid, adding double distilled water to constant volume of 50 mL, placing in refrigerator at 4 deg.C, and storing for 6 months.

Has the beneficial effects that: part of the reagents in the kit provided by the invention are stored by adopting powder, so that the long-term stability of the reagents is ensured, the sample dosage is small (1-5 mu L), the sample pretreatment is simple, the precision of the detection method is good, the repeatability is good, and the accuracy is high; the sample is detected by the enzyme-labeling method, a plurality of samples can be detected at one time by using a 96-pore plate, a large amount of detection time is saved, the larger the sample measurement amount is, the less the time used by a single sample is, and the miniaturization and the kit-making of precision, rapidness, small samples and high flux are realized more easily. Meanwhile, the method provided by the invention has the advantages of simple and rapid reaction, easiness in automation, small dosage of the sample to be detected, low cost of the reagent, easiness in obtaining and more benefit for popularization.

Drawings

FIG. 1 is a graph of the linear range of a standard curve for lactic acid in an example of the present invention;

FIG. 2 is a standard curve for a stability test over one month in an example of the invention, wherein (a) the lactic acid standard curve after one month; (b) a standard curve for lactic acid after three months; (c) standard curve of lactic acid after eight months.

Detailed Description

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, preferred embodiments of the present invention are described below in detail. Specific embodiments of the present invention are given in detail by the following examples.

The embodiment of the invention provides a kit for measuring the content of lactic acid in a biological sample, which consists of a reagent I, a reagent II, a solvent, a lactic acid standard solution, a terminator and a 96-well plate; wherein the content of the first and second substances,

a first reagent: is composed of 23.7 mg Nicotinamide Adenine Dinucleotide (NAD), 10.4 mg Nitrotetrazolium Blue (NBT) and 3.56 mg Phenazine Methosulfate (PMS), powder is stored at-20 ℃ in an X1 bottle;

and a second reagent: consists of 15 mg Lactate Dehydrogenase (LDH), powder is multiplied by 1 bottle and preserved at-20 ℃;

solvent: is composed of a Tris-hydrochloric acid (Tris-HCl) solution with the pH value of 8.8 and a liquid of 100mL multiplied by 1 bottle, and is preserved at the temperature of 4 ℃;

lactic acid standard solution: the concentration of the lactic acid standard solution is 5.56 mmol/L, and the lactic acid standard solution is stored at 4 ℃;

a terminating agent: the liquid is 50 mL x 1 bottles, and the bottles are stored at room temperature;

marking 8 holes of a 96-hole plate as main holes, and respectively adding lactic acid standard solutions with different concentrations; marking other holes as auxiliary holes, and respectively adding the solution to be detected and the other reagents and solvents.

The embodiment of the invention also provides a method for detecting the content of lactic acid in a biological sample, which comprises the following steps:

(1) Preparing a reagent solution:

1) Solvent: 0.1 mol/L Tris-HCl buffer solution pH 8.8: weighing 1.213 g trihydroxymethyl aminomethane, dissolving with double distilled water, adjusting pH to 8.8 with concentrated hydrochloric acid, adding double distilled water to desired volume of 100mL, storing in refrigerator at 4 deg.C for 6 months,

2) Lactic acid standard solution: accurately weighing 5.0 mg lactic acid, dissolving with solvent, metering to 10 mL, preparing into mother liquor with concentration of 5.56 mmol/L, storing in a tight-plugged brown bottle, placing in a refrigerator at 4 deg.C, storing for 6 months,

3) A first reagent: 23.7 mg NAD, 10.4 mg NBT and 3.56 mg PMS are respectively weighed, dissolved by 20 mL solvent, prepared in situ,

4) And a second reagent: accurately weighing 15 mg LDH, dissolving with 15 mL solvent, preparing in situ,

5) A terminating agent: 1.0 Taking 4.2 mL concentrated hydrochloric acid as mol/L hydrochloric acid, using double distilled water to fix the volume to 50 mL, placing in a refrigerator at 4 ℃, and storing for 6 months;

(2) Sample application

1) Selecting 8 wells from a 96-well plate as main wells, adding 5 μ L of lactic acid solution with concentration of 0, 0.56, 1.11, 1.67, 2.22, 2.78, 4.44, 5.56 mmol/L respectively,

2) Selecting other holes as auxiliary holes in a 96-well plate, respectively adding 5 mu L of liquid to be detected,

3) Adding 135 μ L of reagent I and 10 μ L of reagent II into the main hole and each auxiliary hole added with the solution to be detected, mixing well, incubating and reacting at 37 deg.C for 15min,

4) Accurately adding 50 mu L of terminator into each hole in sequence to terminate the reaction;

(3) Detection of

Measuring the absorbance of each well at a wavelength of 550 nm;

(4) Computing

And finally, according to the lactic acid standard in the main hole, using the lactic acid concentration of the serial solutions as an abscissa, using the logarithm of the measured absorbance value A as an ordinate, drawing a standard curve in a semilogarithmic coordinate system, and checking the molar concentration of the lactic acid in the measured sample on the standard curve by using the absorbance value of the solution to be measured in each secondary hole.

Example one

In this example, a mouse liver tissue sample is used as a first test solution, and the following experiment is performed:

(1) Preparation reagent

Solvent: 0.1 mol/L pH8.8Tris-HCl buffer solution,

lactic acid standard solution: 5.56 A standard stock solution of mmol/L lactic acid,

a first reagent: dissolving the powder of the first reagent by using a solvent,

and a second reagent: dissolving the powder of the reagent II by using a solvent,

a terminating agent: 1.0 The mol/L hydrochloric acid is used as the raw material,

PBS solution (pH 7.2): 0.01 mol/L;

(2) Preparing a sample

Accurately weighing the liver tissue according to the weight (g): volume (mL) =1:9, 9 volumes of PBS was added and mechanically homogenized in an ice-water bath. After fully grinding, 7000 r/min, centrifuging for 15 minutes at 4 ℃, taking the supernatant to centrifuge again for 10 minutes at 7000 rpm and 4 ℃ after the supernatant is put in a new EP tube, taking the supernatant to put in the new EP tube, and simultaneously taking part of the supernatant to measure the protein concentration by using a BCA kit;

(3) Sample application and detection

Sample adding and detection analysis operations are carried out according to the table 1;

TABLE 1 operating table for determining lactic acid in sample to be tested

(4) Computing

And drawing a standard curve in a semilogarithmic coordinate system by taking the concentration value of the known lactic acid standard solution as an abscissa and the logarithm of the measured absorbance value A as an ordinate, wherein the regression equation of the standard curve is y =0.0513x +0.0021, and the correlation coefficient R2=0.998. Example one tissue sample was obtained with a lactic acid content of 0.71 + -0.02 μmol/mgprot.

Example two

In this example, the following experiment was performed with the liver cancer cell sample as the second test solution:

(1) Preparation reagent

Solvent: 0.1 mol/L of Tris-HCl buffer solution with the pH value of 8.8,

lactic acid standard solution: 5.56 A standard stock solution of mmol/L lactic acid,

a first reagent: dissolving the powder of the first reagent by using a solvent,

and a second reagent: dissolving the powder of the reagent II by using a solvent,

a terminating agent: 1.0 The mol/L hydrochloric acid is added into the reaction kettle,

cell lysis solution: 50 mM Tris (pH 7.4), 150 mM NaCl,1%, NP-40 (ethylphenylpolyethylene glycol), 0.5% Sodium Deoxycholate (SDS);

(2) Preparing a sample

Extracting a liver cancer cell sample, adding a proper amount of a medium-efficiency lysate according to cell density, centrifuging for 15 minutes at 15000 rpm and 4 ℃ after the cells are completely lysed, taking supernatant liquid in a new EP tube, and simultaneously taking partial supernatant liquid to determine protein concentration by using a BCA kit;

(3) Sample application and detection

Sample adding and detection analysis operations are carried out according to the table 2;

TABLE 2 operating table for determining lactic acid in sample to be tested

(4) Calculating out

Taking the concentration value of the known lactic acid standard solution as an abscissa and the logarithm of the measured absorbance value A as an ordinate, drawing a standard curve in a semilogarithmic coordinate system, wherein the regression equation of the standard curve is y =0.0513x +0.0021, and the correlation coefficient R2=0.998. Example two the content of lactic acid in the cell sample obtained was 31.07. + -. 4.53. Mu. Mol/mgprot.

EXAMPLE III

In this example, a serum (plasma) sample was used as a test solution three, and the following experiment was performed:

(1) Preparation reagent

Solvent: 0.1 mol/L Tris-HCl buffer solution with pH of 8.8,

lactic acid standard solution: 5.56 A standard stock solution of mmol/L lactic acid,

a first reagent: dissolving the powder of the first reagent by using a solvent,

and a second reagent: dissolving the powder of the reagent II by using a solvent,

a terminating agent: 1.0 mol/L hydrochloric acid;

(2) Sample application and detection

Sample adding and detection analysis operations are performed according to table 3;

TABLE 3 lactic acid determination operation table for sample to be tested

(3) Computing

And drawing a standard curve in a semilogarithmic coordinate system by taking the concentration value of the known lactic acid standard solution as an abscissa and the logarithm of the measured absorbance value A as an ordinate. Example III serum samples were obtained with a lactic acid content of 4.51. + -. 0.32 mmol/L.

Taking the third example as an example, the detailed detection data results are as follows:

(1) Linear range: the standard curve equation is y =0.0477x +0.0395, the correlation coefficient R2=0.9913, and the linear range is 0 to 556 mmol/L, as shown in FIG. 1.

(2) Detection limit: and calculating according to a calculation formula of the detection limit to obtain the detection limit of 0.15 mmol/L.

(3) Accuracy: mixing the lactic acid standard solution with the concentration of 1.11 and 2.22 mmol/L with a serum sample, measuring the recovery rate of the method, and calculating the standard addition recovery rate according to a regression equation to be 90.09% -112.61%, wherein the result shows that the method has better recovery rate in Table 4.

TABLE 4 recovery of lactate kit by adding standard

(4) Reproducibility:

4.1 Precisely absorbing 5 mu L of lactic acid standard solution with different concentrations to a 96-well plate, measuring for 3 times, recording the absorbance value, and measuring the RSD to be 1.46-7.08 percent and less than 10 percent, as shown in Table 5, which indicates that the precision meets the requirement.

TABLE 5 lactic acid kit precision experiment

4.2 Take 1.25 μ L of the same serum, measure 6 times of absorbance values in parallel, and measure RSD =0.95%, less than 5%, see table 6, which shows that the test method has good reproducibility and meets the content measurement requirement.

TABLE 6 reproducibility test of lactic acid kit

(5) Stability:

the same serum was used for daily, daytime and monthly stability measurements. In the stability test process during the month, all reagents are stored according to the storage conditions, the lactic acid standard curve needs to be redrawn, and the stability of all test reagents and methods is determined. The detection result shows that the stability of the lactic acid standard curve is good, as shown in figure 2.

The CV in the experimental group is 1.13-3.88% in the day, 3.12% in the daytime and 3.31% in one month; CV was 5.92% in three months; the CV in eight months is 9.51%, and statistical test shows that there is no statistical difference between groups, which proves that the stability of the kit in eight months is good, see Table 7.

TABLE 7 stability test of lactic acid kit in the day, during the day and during the month

(6) Other methods compare:

taking 10 clinical serum samples, and comparing the clinical serum samples with other outsourcing kits; the comparative results are shown in Table 8. The results obtained by the two methods are highly correlated, with a correlation coefficient R2=0.9824.

TABLE 8 comparison of the reagent for this experiment with other kits purchased from outsourcing

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes or modifications made in accordance with the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.

Claims (10)

1. A kit for measuring the content of lactic acid in a biological sample is characterized by comprising a first reagent and a second reagent,

a first reagent: is prepared from nicotinamide adenine dinucleotide, nitrotetrazolium blue and phenazine methyl sulfate, and is stored in powder of 1 bottle at-20 ℃;

and a second reagent: it is composed of lactate dehydrogenase, powder is X1 bottle, and is stored at-20 deg.C.

2. The kit for measuring the content of lactic acid in a biological sample according to claim 1, wherein the mass of nicotinamide adenine dinucleotide contained in the first reagent is 23.7 mg, the mass of nitrotetrazolium blue is 10.4 mg, and the mass of phenazine methosulfate is 3.56 mg.

3. The kit for measuring the content of lactic acid in a biological sample according to claim 1, wherein the mass of the lactate dehydrogenase contained in the second reagent is 15 mg.

4. The kit for measuring the content of lactic acid in a biological sample according to any one of claims 1 to 3, further comprising a solvent, wherein the solvent is composed of a tris-hcl solution, and the solution is stored in a 100mL x 1 bottle at 4 ℃.

5. The kit for measuring the content of lactic acid in a biological sample according to claim 4, wherein the pH of the tris-HCl solution is 8.8.

6. The kit for measuring the content of lactic acid in a biological sample according to any one of claims 1 to 3, further comprising a standard solution of lactic acid, wherein the concentration of the standard solution of lactic acid is 5.56 mmol/L, and the standard solution of lactic acid is stored at 4 ℃.

7. The kit for measuring the content of lactic acid in a biological sample according to any one of claims 1 to 3, further comprising a terminator, wherein the terminator comprises hydrochloric acid, and the solution is stored in a 50 mL x 1 bottle at room temperature.

8. The kit for measuring lactic acid content in a biological sample according to claim 7, wherein the concentration of hydrochloric acid is 1.0 mol/L.

9. A method for measuring the content of lactic acid in a biological sample is characterized by comprising the following steps:

(1) Mixing a biological sample to be detected with a first reagent mainly comprising nicotinamide adenine dinucleotide, nitroblue tetrazolium and phenazine methyl sulfate and a second reagent comprising lactate dehydrogenase to react as follows:

l-lactic acid + nicotinamide adenine dinucleotide

Pyruvic acid + reducing coenzyme I

Reductive coenzyme I + nitrotetrazolium blue

Nicotinamide adenine dinucleotide + purple reduced nitro blue tetrazolium

(2) And detecting the absorbance of the final reactant at 550 nm, and calculating the content of the lactic acid in the sample.

10. The method for measuring the content of lactic acid in a biological sample according to claim 9, wherein the volume of the biological sample to be measured is 1 to 5 μ L, the reaction temperature is 37 ℃, and the reaction time is 15min.

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