CN113552366B

CN113552366B - IV-type collagen determination kit and preparation method thereof

Info

Publication number: CN113552366B
Application number: CN202110768501.4A
Authority: CN
Inventors: 何秋富; 陈壮洪; 邢力策
Original assignee: Zhejiang Yapei Biotechnology Co ltd
Current assignee: Zhejiang Yapei Biotechnology Co ltd
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2022-05-27
Anticipated expiration: 2041-07-07
Also published as: CN113552366A

Abstract

The invention relates to an IV type collagen determination kit and a detection method thereof. The IV type collagen detection kit provided by the invention is used by combining the surfactants, improves the stability of the reagent, simultaneously considers the detection sensitivity, greatly improves the linear range of detection, obviously improves the sensitivity and stability of the detection reagent, is suitable for various full-automatic biochemical analyzers for analysis, and has good adaptability.

Description

IV-type collagen determination kit and preparation method thereof

Technical Field

The invention relates to the technical field of in-vitro diagnostic reagents, in particular to an IV type collagen determination kit and a detection method thereof.

Background

Collagen is a fibrillar glycoprotein, which is a network of one-peptide chains formed by three-strand helices. It has been found that up to 10 kinds of collagen exist in different tissues. Type iv collagen is an important component constituting the basement membrane. The normal intrahepatic basement membrane is mainly present around blood vessels, lymphatic vessels and bile ducts, and is absent from the hepatic sinus wall. The fibrous tissue has life-prolonging life-span along with the development of inflammation in the liver disease, a large amount of collagen is deposited in the fibrous tissue generation process, various collagens are increased, a basement membrane is proliferated, type IV collagen is proliferated firstly in the proliferated collagens, the conversion rate is high, a fibrous clapboard is easy to form, the normal liver lobule structure is damaged, the liver lobules are reconstructed, and false lobules are formed, so that the liver cirrhosis is caused.

The IV type collagen has obvious change in hepatic fibrosis, and when the total collagen amount in mild hepatic injury and hepatic fibrosis is not obviously increased, the content of the serum IV type collagen can be obviously increased, and the change of the content can better reflect hepatic fibrosis and hepatic cell injury and degree.

Diagnosis of early stage hepatic fibrosis or fibrosis of other organs. Type IV collagen is an index mainly used for observing liver cirrhosis, the concentration of the type IV collagen is related to the degree of liver fibrosis, and the degree of liver cirrhosis can be presumed from the concentration of serum type IV collagen.

1. In acute hepatitis, although a large amount of liver cells are destroyed, the concentration of serum IV type collagen has no obvious difference with that of a healthy person because of no obvious connective tissue hyperplasia.

2. The serum IV type collagen of patients with chronic hepatitis, liver cirrhosis and liver cancer is obviously increased, and the increased degree is primary liver cancer, liver cirrhosis, chronic active hepatitis, chronic persistent hepatitis and acute viral hepatitis. At present, the known kit for detecting serum IV type collagen is less, the traditional method has larger defects, the determination sensitivity is insufficient, the operation is complicated, the accuracy is low and the like, and the kit is not beneficial to clinical popularization and use.

At present, IV type collagen detection reagents with high sensitivity and strong specificity, in particular to automatic detection reagents with good quality, are lacked in the market. The existing IV type collagen determination method mainly uses a time-resolved fluorescence immunochromatography method, a chemiluminescence immunoassay method and the like. The time-resolved fluorescence immunochromatography method has good stability but low sensitivity; the chemiluminescence method has high sensitivity, but the reagent has poor stability, needs expensive special chemiluminescence detection equipment, is not beneficial to development in a conventional laboratory, and has obvious limitation on clinical application. The existing latex immunoturbidimetry is simple and rapid to operate, but has low sensitivity and narrow linear range. Therefore, it is urgently needed to develop a type IV collagen detection kit with high sensitivity, strong specificity, simple detection method and wide application range.

Disclosure of Invention

According to previous studies by the inventors, it was found that there is a clear correlation between surfactants and antibodies binding to type IV collagen. In order to overcome the defects in the prior art, the invention provides a type IV collagen determination kit, which comprises a surfactant, a stabilizer, a buffer solution, a type IV collagen antibody and an antifreeze agent which are combined.

In one embodiment, the surfactant of the present invention is a combination of F88 and polyoxyethylene lauryl ether, preferably, the ratio of F88 to polyoxyethylene lauryl ether is 1: 1.

In one embodiment, the stabilizing agent in the present invention is one or more selected from inorganic salts, preservatives, and cell protective agents;

in one embodiment, the inorganic salt in the present invention is one or more of sodium chloride, potassium chloride or magnesium chloride, preferably, the inorganic salt in the present invention is sodium chloride;

in one embodiment, the preservative in the present invention is one of Proclin 300, benzoic acid, sodium benzoate, and sodium azide, and preferably, the preservative in the present invention is Proclin 300;

in one embodiment, the buffer solution in the present invention is one or more of a phosphate buffer solution, a tris (hydroxymethyl) aminomethane sulfonic acid (TAPS) buffer solution, and an acetate buffer solution, and preferably, the buffer solution in the present invention is a phosphate buffer solution;

in one embodiment, the cytoprotective agent of the present invention is one or more selected from ovalbumin, bovine transferrin, human serum albumin, bovine serum albumin and hemocyanin, and preferably, the cytoprotective agent of the present invention is bovine serum albumin.

In one embodiment, the collagen type iv antibodies of the invention are derived from an animal or a recombinant antibody, such as rabbit, sheep, chicken, camel, mouse, rat. Most preferably, the collagen type IV antibody of the invention is a rabbit anti-human collagen type IV antibody.

In one embodiment, the collagen iv antibody may be selected from collagen iv antibody latex microsphere particles, and more preferably, the coupling agent used for coupling the antibody and the latex microsphere is a combination type coupling agent, and the combination type coupling agent comprises the following components: the mass ratio of the carbodiimide to the O-benzotriazole-N, N, N' -tetramethylurea tetrafluoroborate to the O-benzotriazole-tetramethylurea hexafluorophosphate is 1.2: 1.

In one embodiment, the antifreeze of the present invention is one or more of methanol, ethanol, ethylene glycol, water soluble amides and calcium chloride, brine, and the like. Preferably, the antifreeze agent in the present invention is ethylene glycol.

In a preferred embodiment, the kit of the invention comprises at least reagent 1 and reagent 2:

reagent 1

Reagent 2

The invention has the following beneficial effects:

1. the preparation steps of specific substances in the kit are simple, the cost is low, and the kit is more suitable for industrial production and clinical popularization and application;

2. the applicant selects the surfactant F88 and the polyoxyethylene lauryl ether to be added in the kit by the combined screening of the surfactants, thereby obviously improving the analysis sensitivity and precision of the kit and enlarging the linear range of detection.

3. The glycol is added into the kit, so that the freezing resistance of the kit is improved, and the kit can be transported or stored at a low temperature and still maintain good detection performance.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example 1: plotting of calibration curves

Reagent 1

Reagent 2

Reagent 1, reagent 2 and collagen calibrator were packaged into a kit.

Setting parameters on Hitachi-7180 full-automatic biochemical analyzer:

measurement mode	End point method	Sample size	6μl
				Reagent 1	180μl	Reagent 2	60μl
Measuring temperature	37℃	Wavelength of light	600nm
				Optical path	1cm	Reaction direction	Is just

Procedure for the preparation of the

Calibration procedure

And calibrating by using an IV type collagen calibrator, performing multi-point calibration, and adopting a nonlinear calibration mode such as Logit, Spline and the like.

Calculation of results

And (5) five-point calibration, namely taking purified water from the first tube, and inputting a value of 0.0ng/ml, and taking a calibration product from the second tube to the fifth tube, and inputting a corresponding value. And making a dose/response curve according to the absorbance and the value of the calibrator, and calculating the content of the sample on the dose/response curve according to the absorbance value.

Example 2: determination of the Linear Properties

And (3) carrying out equal-difference mixed dilution on a certain sample (serum) with high IV type collagen concentration and a certain sample with low IV type collagen concentration, measuring the diluted concentration, measuring for three times, calculating the average value, comparing with the theoretical concentration, and calculating the linear deviation of the sample. It can be seen that the linear range of the kit can reach 30-420 ng/ml.

Linear result

Example 3: precision and recovery

Serum samples at high, medium and low concentrations (concentrations of 90, 180 and 360ng/ml, respectively) were assayed in 20 replicates and the assay data and data analysis are shown in the following table.

TABLE 5 precision and recovery

The latex immunoturbidimetry assay kit has the advantages of high accuracy, high recovery rate of 85-115%, high precision and no CV exceeding 10.0%.

Example 4 (comparative):

the validation was carried out using a combination of F88+ triton x100, and F88 single surfactant as a comparative example:

comparative example 1:

reagent 1

Reagent 2

Reagent 1, reagent 2 and collagen calibrator were packaged into a kit.

Comparative example 2:

reagent 1

Reagent 2

Reagent 1, reagent 2 and collagen calibrator were packaged into a kit.

Linear results using a combination of F88 and TritonX100

Linear results using F88 alone

After calibration according to the procedure of example 1, the measurement, experimental parameters and samples for the linear range were identical to those of example 2, except that the reagents used were different: the results show that the linear range of the kit is only 30-330ng/ml (using F88+ TritonX100), 30-250ng/ml (using F88 alone), much lower than the technical effect of the combination of F88 and polyoxyethylene lauryl ether, respectively, when a single surfactant is used.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The IV-type collagen determination kit is characterized by comprising a reagent 1 and a reagent 2, wherein the reagent 1 and the reagent 2 are prepared according to the following mixture ratio:

reagent 1

Disodium hydrogen phosphate 1.09 g/L

Sodium dihydrogen phosphate 0.32 g/L

Sodium chloride 9 g/L

PEG 6000 5g/L

Proclin 300 1g/L

Ethylene glycol 10g/L

Reagent 2

Disodium hydrogen phosphate 1.09 g/L

Sodium dihydrogen phosphate 0.32 g/L

Rabbit anti-human type IV collagen antibody 2g/L

Proclin300 1g/L

F88 2g/L

Polyoxyethylene lauryl ether 2g/L

Ethylene glycol 10g/L

BSA 2g/L。