CN113433064A - Hyaluronic acid detection kit and method thereof - Google Patents

Abstract

The invention discloses a kit for detecting hyaluronic acid and a method thereof. The invention is used for detecting the content of hyaluronic acid in serum under a certain wavelength after the HABP is coated on the latex microspheres and the kit is sealed by using a sealing agent. The experimental result shows that the hyaluronic acid detection kit prepared by the latex immunoturbidimetry method has better accuracy, precision, linearity, stability and sensitivity than hyaluronic acid detection kits of other methodologies, and the method has better safety, fast detection speed and easy automation compared with other methods, and is effectively popularized and applied in industry on trial.

Description

Hyaluronic acid detection kit and method thereof

Technical Field

The invention relates to the technical field of in-vitro diagnostic reagent biochemical reagents, in particular to a hyaluronic acid detection kit and a method thereof.

Background

Hyaluronic acid (hyaluronan HA) is a macromolecular polysaccharide whose main site of synthesis is mesenchymal cells and enters the human blood through lymphatic circulation, where it is metabolized in the liver and discharged after glomerular filtration. Normal human serum generally contains HA in an amount of less than 100 ng/ml. The hyaluronic acid is absorbed and degraded by the liver endothelial cells, so that the absorption and degradation functions of the hyaluronic acid of patients with liver diseases, particularly patients with liver cirrhosis are reduced due to the damage of the liver endothelial cells, so that the content of the hyaluronic acid in the serum of the patients with liver diseases is increased, and the change of the content of the hyaluronic acid in the serum can be used as an index for early diagnosis of liver fibrosis. In addition, in the case of rheumatoid arthritis and scleroderma, the content of hyaluronic acid in serum is increased by increasing the source of hyaluronic acid; or liver and kidney diseases can affect the discharge of hyaluronic acid in human body, so that the content of hyaluronic acid in serum is increased. Therefore, the change of the content of hyaluronic acid in the serum can be detected to reflect the pathological changes of the liver and the degree of cirrhosis of the liver.

The existing detection methods for the content of hyaluronic acid in serum mainly comprise radioimmunoassay, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA). The radioimmunoassay is mainly that a certain amount of Hyaluronic Acid Binding Protein (HABP) is added into a sample with unknown HA amount; adding iodine to mark hyaluronic acid to combine with HABP left from specimen reaction; and then using a first antibody, namely sheep or rabbit anti-HABP serum, and a second antibody, namely donkey anti-sheep or sheep anti-rabbit serum, to precipitate the HABP, determining the radioactivity of the HABP, wherein the iodine-labeled HA combined with the HABP is in negative correlation with the HA content in the sample, using an HA standard substance to synchronously compare and operate to make a radioactive concentration standard curve, and finding out a corresponding concentration value of the measured radioactive relative value of the sample on the standard curve. However, the method has high requirements on equipment and personnel, and is not beneficial to application and popularization. The ELISA detection of HA is mainly based on HABP (hyaluronic acid binding protein) as an antibody and a double-sandwich method, and compared with an radioimmunoassay, the detection sensitivity is similar, but the HA value of serum is obviously lower than that of the radioimmunoassay. The chemiluminescence immunoassay method is mainly based on two technologies of competition and sandwich, the principle of the competition method is to combine HA in a sample with coated HA on a solid phase carrier and HABP in a diluent to realize detection, and the higher the HA concentration of the sample is, the lower the luminous value is; the sandwich method realizes detection by forming a solid phase coated HABP-HA-HABP sandwich compound, and the luminous value is in direct proportion to the HA concentration of a sample. However, the content of HA in the serum of normal human detected by the method and the enzyme-immune and radioimmunoassay is very different, and the analysis probably results from that HA is a polysaccharide molecule with uneven molecular weight, the molecular weight range is 105-107, the double-antibody sandwich method needs more than 20 glycosyl groups to be combined with protein to detect the HA, and the radioimmunoassay and the enzyme-immune method do not need more than 20 glycosyl groups in the HA, so the detection range is wider than that of the double-antibody sandwich method. The detection of hyaluronic acid by enzyme immunization and radioimmunoassay has the defects.

Disclosure of Invention

In view of this, the present invention aims to provide a method for detecting the content of hyaluronic acid in a serum sample, which is simple and convenient to operate, low in cost, capable of being automated, and high in accuracy, so that the method can be widely applied clinically.

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

a hyaluronic acid detection kit comprising reagents R1, R2 and a calibrator; wherein, the reagent R1 comprises buffer solution, sodium chloride, preservative and stabilizing agent. The reagent R2 comprises an activation buffer, a coating buffer, a blocking solution, a storage buffer, latex microspheres and HABP, and a coupling agent.

The detection principle of the invention is as follows: hyaluronic Acid Binding Protein (HABP) is coated on latex microspheres through a coupling agent, after HA in a sample is specifically bound with the HABP on the microspheres, the content of HA in a serum sample is quantitatively measured through measuring the change of absorbance under a certain wavelength, wherein the change is proportional to the content of HA in the sample.

The concentration of the R1 component of the reagent is as follows: buffer solution 0.1-100mmol/L, sodium chloride 1-20g/L, preservative 0.1-10g/L, stabilizer 0.1-10 g/L; the component concentration of the reagent R2 is as follows: 0.1-100mmol/L of activation buffer solution, 0.1-100mmol/L of coating buffer solution, 0.1-100mmol/L of confining liquid, 0.1-100mmol/L of storage buffer solution, 1-100ml/L, HABP 2-200ml/L of latex microspheres, 10.1-5 g/L of coupling agent and 20.05-2.5 g/L of coupling agent.

The preferred concentrations of the components of the reagent R1 according to the invention are: 25mmol/L buffer solution, 9g/L sodium chloride, 5g/L preservative and 5g/L stabilizer; the preferred concentrations of the reagent R2 components are: 50mmol/L of activation buffer solution, 50mmol/L of coating buffer solution, 25mmol/L of blocking solution, 50mmol/L of storage buffer solution, 30ml/L, HABP 50ml/L of latex microspheres, 11 g/L of coupling agent and 20.5 g/L of coupling agent.

In the preferable reagent R1, the buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution; in the reagent R2, the activating buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, the coating buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, the confining solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, bovine serum albumin is added into the confining solution, and the storage buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution.

Preferably, the pH of the buffer solution in the reagent R1 and the reagent R2 is 5.0-9.0.

In order to improve the stability of the kit of the invention, the preferred stabilizers in the reagents R1 and R2 are the PEG series. The preservatives in reagents R1 and R2 are sodium azide, sodium nitrite, Proclin series, preferably Proclin 300. The coupling agents in reagent R2 were N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC). The latex microspheres in reagent R2 had particle sizes of 0.08-0.5. mu.m.

The invention also provides a detection method of the hyaluronic acid detection kit, which comprises the following steps:

(1) sequentially adding the reagent R1 and a sample to be detected, uniformly mixing, incubating for 4-10 minutes at 37 ℃, then adding the reagent R2, uniformly mixing, incubating for 4-10 minutes at 37 ℃, and then measuring the absorbance at the wavelength of 600 nm;

(2) and calculating the concentration of the hyaluronic acid in the sample to be detected according to the calibrated standard curve.

Preferably, the standard curve is calibrated by an HA calibrator, and the concentration of the HA calibrator is 0-1000 ng/ml.

The kit disclosed by the invention can be used for specifically binding HA in a serum sample by coating the natural protein HABP on the latex microspheres. The invention determines the HA content in the serum. The test result shows that the hyaluronic acid detection kit has the same measurement result with the radioactive immunoassay method, is more accurate than the measurement result of enzyme immunoassay and chemiluminescence reagents, has good stability, can be stably stored for at least 13 months in a refrigeration state, has wide linear range, high sensitivity and high detection speed, and is easy for automatic operation.

Drawings

FIG. 1 is a line graph showing the hyaluronic acid assay kit A of the present invention.

FIG. 2 is a line graph showing the hyaluronic acid assay kit B of the present invention.

FIG. 3 is a line graph showing the hyaluronic acid assay kit C of the present invention.

Detailed Description

The embodiment of the invention discloses a hyaluronic acid detection kit and a method. The technical means, advantages and effects of the present invention are further illustrated by the following examples, which are intended to illustrate the present invention but not to limit the scope of the present invention, and those skilled in the relevant art can change and modify the examples without departing from the spirit and scope of the present invention, but such changes and modifications are within the scope of the present invention.

Example 1

The preparation process of the kit for detecting hyaluronic acid in serum comprises the following steps:

composition and concentration of reagent R1:

MES(pH6.0) 25mmol/L

sodium chloride 9g/L

PEG6000 5g/L

Proclin300 5g/L

Composition and concentration of reagent R2:

composition and concentration of the activation buffer:

MES(pH6.0) 50mmol/L

Proclin 300 5g/L；

composition and concentration of coating buffer:

HEPES(pH7.5) 50mmol/L

Proclin300 5g/L

composition and concentration of blocking buffer:

glycine (pH7.0) 25mmol/L

BSA 5g/L

Proclin300 5g/L

Composition and concentration of storage buffer:

Tris-HCl(pH7.0) 50mmol/L

Proclin300 5g/L

latex microspheres 30ml/L

HABP 50ml/L

NHS 1g/L

EDC 0.5g/L

Preparation process of reagent R1:

sequentially adding MES, sodium chloride, PEG6000 and Proclin300 compounds with preparation amount and purified water with preparation amount of 80%, adding another substance after each substance is completely dissolved, adjusting pH to 6.0 with 1N or 6N NaOH, and adding purified water to constant volume to preparation amount.

Preparation process of reagent R2:

(1) adding the prepared latex microspheres into the prepared activation buffer solution, uniformly mixing, sequentially adding the prepared NHS and EDC, reacting in a constant temperature oscillator at 37 ℃ for 30 minutes, and taking out;

(2) adding the HABP raw material with the preparation amount into the coating buffer solution with the preparation amount, and uniformly mixing;

(3) adding the solution in the step (2) into the solution in the step (1), uniformly mixing, placing the mixture in a constant temperature oscillator at 37 ℃ for reacting for 2 hours, and taking out;

(4) adding the prepared confining liquid into the step (3), uniformly mixing, placing the mixture into a constant-temperature oscillator at 37 ℃ for reaction for 30 minutes, and taking out;

(5) adding the prepared storage buffer solution into the reagent (4), and uniformly mixing to obtain the reagent R2.

The application of the hyaluronic acid detection kit in serum in a full-automatic biochemical analyzer is as follows:

the analysis method comprises the following steps: velocity method

The reaction direction is as follows: ascending reaction

The calibration method comprises the following steps: spline

Measurement of dominant wavelength: 600nm

Measurement of sub-wavelength: 800nm

Measuring temperature: 37 deg.C

The sample adding amount proportion is as follows: sample preparation: r1: R2 ═ 2:180:60(μ L)

The method comprises the following operation steps: mu.L of the test sample was added to 180. mu.L of the reagent R1, incubated at 37 ℃ for 3 to 5 minutes and then the absorbance A1 was read, then the reagent R260. mu.L was added, mixed well, incubated at 37 ℃ for 5 minutes and then the absorbance A2 was read, and Δ A ═ A2-A1 was calculated.

Calibration: adopting a multi-point calibration method, and detecting by using a Toshiba-40 full-automatic biochemical analyzer, wherein the concentration of a calibrator is as follows: c10 ng/mL, C250 ng/mL, C3100 ng/mL, C4200 ng/mL, C5400 ng/mL, C61200 ng/mL.

Example 2

Hyaluronic acid assay kit performance evaluation

First, analysis sensitivity

The three kits A, B, C of the present invention, formulated in example 1, were simultaneously calibrated and the results are shown in Table 1.

TABLE 1 calibration results of the three-batch hyaluronic acid determination kit A, B, C of the present invention

As can be seen from Table 1, the absorbance change rates of the kit A, B, C of the present invention were 0.0258, 0.0255, and 0.0269, respectively, each of which was greater than 0.005, at a hyaluronic acid content of 50 ng/L.

Second, accuracy determination

The accuracy was determined by recovery experiments with the three-batch kit A, B, C of the invention formulated in example 1.

A certain volume of standard solution is added into a clinical serum sample, and the recovery rate is calculated through measurement. The measurement results and the recovery rate are shown in Table 2.

TABLE 2 determination results of the accuracy of three batches of hyaluronic acid determination kits of the present invention

As can be seen from Table 2, the recovery rate of the kit A, B, C of the invention is from 104.67% to 99.80%, and is less than 105% -95%, which indicates that the accuracy of the kit is high.

Third, accelerated stability detection

A batch of kit A of the invention prepared in example 1 was accelerated through a water bath at 37 ℃ and then calibrated and 10 fresh serum samples were assayed. The measurement results are shown in tables 3 and 4.

TABLE 3 accelerated calibration results of hyaluronic acid assay kit

TABLE 4 hyaluronic acid assay kit accelerated post-assay results for 10 fresh serum samples

Unit: ng/L

As can be seen from Table 4, the deviation of the mean value of 10 fresh serum samples measured by the kit A of the invention is less than 5% after accelerating for 3 days, 5 days, 7 days and 10 days at 37 ℃, and the performance is stable. It can be seen that the kit A of the invention has better accelerated stability at 37 ℃.

Fourth, batch to batch experiment

The same control samples were assayed in triplicate at each level using the three kits A, B, C of the invention prepared in example 1, and the results are shown in Table 5.

TABLE 5 results of inter-batch Difference experiment of three hyaluronic acid assay kits A, B, C of the present invention

As can be seen from Table 5, the kit A, B, C of the present invention measured the interrun difference of low-value quality control to be 0.36%, and the interrun difference of high-value quality control to be 0.15%. As can be seen, the kit had less than 5% batch-to-batch variation, which was very small.

Fifth, Linear detection

Three batches of the kit A, B, C of the present invention prepared in example 1 were subjected to a linear assay by measuring a series of concentration gradient standard solutions prepared from hyaluronic acid concentrate. The linearity results are shown in Table 6, Table 7, Table 8, FIG. 1, FIG. 2 and FIG. 3.

TABLE 6 hyaluronic acid assay kit A Linear results

TABLE 7 hyaluronic acid assay kit B Linear results

TABLE 8 hyaluronic acid assay reagent C linearity results

As can be seen from tables 6, 7, 8, 1, 2 and 3, the hyaluronic acid assay kit A, B, C of the present invention showed linear correlation coefficients R2 of 0.9999, [15.63, 250] ng/L, with absolute deviation less than. + -. 5 ng/L; within the range of (250, 1000] ng/L, the relative deviation is less than +/-2%.

Claims (12)

1. The hyaluronic acid detection kit is characterized by comprising reagents R1 and R2 and a calibrator, wherein the reagent R1 comprises a buffer solution, sodium chloride, a preservative and a stabilizer; reagent R2 contains activation buffer, coating buffer, blocking solution, storage buffer, as well as latex microspheres and HABP, and coupling agent.

2. The hyaluronic acid detection kit of claim 1, wherein the reagent R1 component concentration is: buffer solution 0.1-100mmol/L, sodium chloride 1-20g/L, preservative 0.1-10g/L, stabilizer 0.1-10 g/L; the component concentration of the reagent R2 is as follows: 0.1-100mmol/L of activation buffer solution, 0.1-100mmol/L of coating buffer solution, 0.1-100mmol/L of confining liquid, 0.1-100mmol/L of storage buffer solution, 1-100ml/L, HABP 2-200ml/L of latex microspheres, 10.1-5 g/L of coupling agent and 20.05-2.5 g/L of coupling agent.

3. The hyaluronic acid detection kit of claim 2, wherein the reagent R1 component concentration is: 25mmol/L buffer solution, 9g/L sodium chloride, 5g/L preservative and 5g/L stabilizer; the component concentration of the reagent R2 is as follows: 50mmol/L of activation buffer solution, 50mmol/L of coating buffer solution, 25mmol/L of blocking solution, 50mmol/L of storage buffer solution, 30ml/L, HABP 50ml/L of latex microspheres, 11 g/L of coupling agent and 20.5 g/L of coupling agent.

4. The hyaluronic acid detection kit of claims 1-3, wherein in the reagent R1, the buffer is one or more of phosphate buffer, Tris-HCl, MES buffer, HEPES buffer, and glycine buffer; in the reagent R2, the activating buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, the coating buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, the confining solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution, bovine serum albumin is added into the confining solution, and the storage buffer solution is one or more of phosphate buffer solution, Tris-HCl, MES buffer solution, HEPES buffer solution and glycine buffer solution.

5. The hyaluronic acid detection kit of claim 4, wherein the pH of the buffer solution in reagents R1 and R2 is 5.0-9.0.

6. The hyaluronic acid detection kit of claims 1-3, wherein the stabilizing agent in reagent R1 is PEG series.

7. The hyaluronic acid detection kit according to claims 1-3, wherein the preservative in the reagent R1 is sodium azide, sodium nitrite, Proclin series.

8. The hyaluronic acid detection kit of claims 1-3, wherein the coupling agent in reagent R2 is N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC).

9. The hyaluronic acid detection kit of claims 1-3, wherein the particle size of the latex microspheres in reagent R2 is 0.08-0.5 μm.

10. The method for detecting hyaluronic acid detection kit according to claims 1-9, comprising the steps of:

(1) sequentially adding the reagent R1 and a sample to be detected, uniformly mixing, incubating for 4-10 minutes at 37 ℃, then adding the reagent R2, uniformly mixing, incubating for 4-10 minutes at 37 ℃, and then measuring the absorbance at the wavelength of 600 nm;

(2) and calculating the concentration of the hyaluronic acid in the sample to be detected according to the calibrated standard curve.

11. The method of claim 10, wherein the standard curve is calibrated with a hyaluronic acid calibrator.

12. The method of claim 11, wherein the concentration of the hyaluronic acid calibrator is 0-1000 ng/mL.

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