CN101368970A

CN101368970A - Chemical luminescence immune assay determination reagent kit for detecting Beta-2 microglobulin

Info

Publication number: CN101368970A
Application number: CNA2008101054198A
Authority: CN
Inventors: 姚洪涛; 应希堂; 宋胜利; 胡国茂; 郑金来; 于尚永
Original assignee: KEMEI DONGYA BIOLOGICAL TECHNOLOGY Co Ltd BEIJING
Current assignee: BEIJING KEMEI BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2008-04-29
Filing date: 2008-04-29
Publication date: 2009-02-18

Abstract

The invention discloses a test kit and a preparing method thereof, which uses chemiluminescence immunoassay analysis method to quantitatively detect Beta-2 microglobulin in blood. The quantitative test kit of detecting Beta-2 microglobulin through the chemiluminescence immunoassay analysis method comprises fluorescein antibody coated solid-phase carrier, compound of fluorescein-labeled capture antibody and enzyme-labeled detection antibody, chemiluminescence zymolyte on which the enzyme reacts, and Beta-2 microglobulin series standard sample. The test kit can be applied to open type semi-automatic chemiluminescence tester, and can also be used in automatic measuring system, and can ensure mass swift detection, low cost and easier popularization.

Description

Chemiluminescence immunoassay determination kit for detecting Beta-2 microglobulin

Technical Field

The invention relates to the medical field of immunoassay, and particularly provides a quantitative determination kit for a chemiluminescence immunoassay Beta-2 microglobulin and a preparation method thereof.

Background

Beta-2 microglobulin (Beta 2-MG), a low molecular weight (MW11800) globulin which was first isolated from urine of patients with renal tubular disease in 1968 by Berggard, all nucleated cells can be produced, are mainly produced by lymphocytes, are widely present in human plasma, urine, cerebrospinal fluid and other body fluids and neutralize the surface of nucleated cell membranes, under normal conditions, Beta-2 microglobulin on the cell surface is repeatedly combined and dissociated with Beta-2 microglobulin free in body fluid to maintain a dynamic equilibrium state, in certain pathological conditions, such as lung cancer, kidney cancer, breast cancer, and digestive system malignancies, this dynamic equilibrium is disrupted, and the levels of free Beta-2 microglobulin in the blood are abnormally elevated, therefore, in recent years, Beta-2 microglobulin has become an important diagnostic marker for tumor serology and is widely concerned.

The current clinical diagnosis technology of Beta-2 microglobulin mainly comprises the following steps: radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), Chemiluminescence (CLIA), and the like, but these methods all have various degrees of disadvantages: the radio-immunity is a method which is commonly used in clinic, has the advantages of accurate result, wide linear range and complex and long operation time, and radioactive markers can cause harm to operators and environmental pollution, and is gradually replaced by other methods at present. ELISA has the defects of large difference of results among methods, narrow linear range and the like.

The chemiluminescence immune analysis is a new immune analysis technology developed after fluorescence, radioactive isotope and enzyme immune analysis, according to a large amount of experimental results and clinical application data, from the aspects of practicability, stability, accuracy and development prospect, the chemiluminescence immune analysis is in the leading position in the non-radioactive labeling analysis technology, represents the direction and trend of the current world development, not only has the specificity of immune reaction, but also has high sensitivity of chemiluminescence reaction (the detection limit can reach 10)^-15～10^-18mol/L). The chemiluminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, long effective period, safety, no toxicity, no pollution and the like, and becomes the first choice for replacing the radio immunoassay and enzyme immunoassay technology.

Chemiluminescence enzyme immunoassay is to perform immunoreaction with enzyme-labeled bioactive substances (such as enzyme-labeled antigen or antibody), to obtain luminous substrate, to emit light under the action of signal reagent, and to perform luminous measurement with luminous signal detector. The currently commonly used labeling enzyme is horseradish peroxidase (HRP), the commonly used substrate of HRP is luminol (3-aminophthalic hydrazide, luminol) or a derivative thereof, such as isoluminol (4-aminophthalic hydrazide) and the like, the oxidation reaction of the luminol is carried out in an alkaline buffer, in the presence of peroxidase and active oxygen, an excited state intermediate is generated, and when the excited state intermediate returns to a ground state, the excited state intermediate emits light, and the wavelength of the excited state intermediate light is 425 nm. The intensity of luminescence depends on the concentration of the enzyme in the enzyme immunoreactant.

Based on the specific high affinity between fluorescein and fluorescein antibody, there are generally two modes of use: 1. the method uses fluorescein labeling to detect the anti/protomer, fluorescein antibody labeling enzyme, further enhances reaction signals on the basis of double antigens or double antibody sandwich, the mode is gradually developed and applied in biology, at present, commercial reagents are available, and related labeling programs are basically solidified, and the conditions greatly facilitate the application of the method; 2. the method is characterized in that a fluorescein antibody is coated on a solid phase, and the fluorescein is used for marking a capture antigen/antibody as an indirect coating mode of the capture antibody, so that the possibility is provided for the solid phase of antigens/antibodies which are not easy to be directly solidified, meanwhile, due to the good stability of the fluorescein and the fluorescein antibody, the application range of the antigens/antibodies which are directly solidified and have unstable properties is expanded, but the mode is less in application at present and has large development space.

Disclosure of Invention

The invention creatively applies the chemiluminescence immunoassay and the fluorescein/fluorescein antibody system to the quantitative detection of the Beta-2 microglobulin, can not only keep the high sensitivity of the chemiluminescence immunoassay, but also make up the defects that the Beta-2 microglobulin is not easy to coat and is easy to inactivate after coating by utilizing the advantages of the fluorescein/fluorescein antibody system. In addition, the existing chemiluminescence immunoassay reagent is a closed full-automatic chemiluminescence measuring system, and an expensive full-automatic chemiluminescence measuring instrument is needed, so that the popularization and the use are limited, and the reagent cannot be widely applied to clinical diagnosis and scientific research work. The kit adopts a microplate chemiluminescence immunoassay technology, can be applied to an open semi-automatic chemiluminescence measuring instrument, can also be applied to a full-automatic measuring system, can realize large-batch quick detection, and is low in use cost and easier to popularize and apply.

The invention aims to provide a kit for quantitatively detecting Beta-2 microglobulin in blood by combining a fluorescein/fluorescein antibody technology with a chemiluminescence immunoassay method, and the invention also aims to provide a preparation method of the kit

The kit for quantitatively detecting Beta-2 microglobulin in blood by using a chemiluminescence immunoassay method comprises the following components:

1. solid phase carrier coated with fluorescein antibody

2. Fluorescein-labeled capture antibodies

3. Enzyme-labeled detection antibody

4. Luminescent substrates required for the action of the above enzymes

5. Beta-2 microglobulin series standard, and

6. concentrated washing liquid

Specifically, the preparation process of the kit comprises the following steps:

A. preparation of solid phase carrier: selecting high-purity and high-affinity fluorescein antibody, diluting to a certain concentration by using a coating buffer solution, coating a solid phase carrier by a certain amount, sealing by using a sealing solution, airing, adding a drying agent, and sealing by using an aluminum foil bag.

B. Fluorescein labeling of capture antibody: after a high-purity high-affinity Beta-2 microglobulin capture antibody is selected and dialyzed by using a fluorescein labeled buffer solution, a certain amount of high-quality commercial fluorescein reagent is added, after a certain time of reaction, the high-purity high-affinity Beta-2 microglobulin capture antibody is dialyzed by using the fluorescein labeled buffer solution again, and the high-purity high-affinity Beta-2 microglobulin capture antibody is stored at minus 20 ℃ for later use.

C. Enzyme labeling of the detection antibody: selecting a Beta-2 microglobulin detection antibody which has high purity and high affinity and is strictly matched with the selected capture antibody, dialyzing by using an enzyme labeling buffer solution, adding a certain amount of activated enzyme, reacting for a certain time in a dark place, precipitating by using a saturated ammonium sulfate solution, centrifuging, redissolving the precipitate by using a phosphoric acid buffer solution, adding isometric glycerol, and keeping at-20 ℃ for later use.

D. Mixing a fluorescein labeled capture antibody and an enzyme labeled detection antibody: before the kit is assembled, the capture antibody marked by fluorescein and the detection antibody marked by enzyme are diluted by enzyme diluent in proportion and then are subpackaged according to the required amount.

E. Preparation of luminescent substrate: based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value is 8.0-10.0, and the chemiluminescent substrate A solution is prepared and then subpackaged according to the required amount;

based on 1000mL of the chemiluminescent substrate B solution, the chemiluminescent substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20, 51.58g of Na2HPO 4.12H 2O and 8.74g of NaH2PO 4.2H 2O, the pH value is 7.0-7.6, and the chemiluminescent substrate B solution is prepared and then subpackaged according to the required amount.

F. Preparing Beta-2 microglobulin series standard products: commercial high-purity Beta-2 microglobulin is selected, the concentration of the microglobulin is determined by national standard substance contrast measurement, and calf serum is used for serial dilution: s6: 8.0. mu.g/ml, S5: 4.0. mu.g/ml, S4: 2.0. mu.g/ml, S3: 1.0. mu.g/ml, S2: 0.5. mu.g/ml, S1: 0.2. mu.g/ml, S0: 0 calf serum.

G. Preparation of concentrated washing solution: based on 1000mL of the concentrated washing solution, the concentrated washing solution comprises 4g of NaH2PO 4.2H2O, 58g of Na2HPO 4.12H2O, 175.32g of NaCl, 10mL of Tween-20 and 1mL of Proclin-300, the pH value of the concentrated washing solution is 7.0-7.6, and the concentrated washing solution is prepared and then is divided into two parts according to the requirement.

H. And assembling to obtain the finished product kit.

Wherein,

in the step A, the solid phase carrier is a 96-well plate or a plastic tube, and the coating buffer solution is a carbonic acid buffer solution with pH9.6, a phosphate buffer solution with pH7.2 or a citric acid buffer solution with pH 4.7; the concentration is 1-5 mug/ml; the coating amount is 100-; the coating condition is 4 ℃ overnight or room temperature for 6 hours; the blocking amount is 150-; the blocking conditions were either 37 ℃ for 2 hours or room temperature for 4 hours. The preferable scheme is as follows: 96-well plates, diluted 2.5. mu.g/ml in carbonate buffer, 200. mu.l/well, coated overnight at 4 ℃ and blocked at 37 ℃ for 2 hours at 250. mu.l/well.

In the step B, the buffer solution for labeling the fluorescein is phosphate buffer solution or carbonic acid buffer solution, the amount of the fluorescein is 15-50 mu g/mg of the antibody, the reaction time is 1-4 hours, the preferable scheme is 20mMpH7.2 of phosphate buffer solution, the mass ratio of the fluorescein to the antibody is 30 mu g/mg, the reaction time is 2 hours, and the same volume of glycerol is added for preservation at-20 ℃.

In the step C, the selected enzyme is Alkaline Phosphatase (AP) or horseradish peroxidase (HRP), the labeling buffer solution is phosphate buffer solution (AP) or carbonate buffer solution (HRP), the coupling agent used by the AP can be carbodiimide (EDC) or glutaraldehyde, the activating agent used by the HRP is sodium periodate, the preferred scheme is that the HRP is labeled by a modified sodium periodate method, and after labeling, glycerol with the same volume is added, and the HRP is stored at-20 ℃ for later use.

In step D, the dilution of the fluorescein-labeled capture antibody may be 1: 2,000-1: 8,000, the dilution of the enzyme-labeled detection antibody may be: 1: 5,000-1: 20,000, enzyme dilutions used were: 20mMPBS, pH7.4, 1-4% BSA, 1-3% sucrose, 0.1-1.0% gelatin, 0.05-0.1% edible fuchsin, 0.05-0.2% biological preservative. Preferably, we select dilutions of fluorescein-labeled capture antibody as follows: 1: 5,000, dilutions of enzyme-labeled detection antibody: 1: 10,000, enzyme dilutions used were: 20mM PBS, pH7.4, 1.5% BSA, 2.5% sucrose, 0.5% gelatin, 0.4% edible fuchsin, 0.1% biological preservative, and the subpackage amount is 12 ml/bottle.

In the step E, the subpackaging amount of the substrate A liquid and the substrate B liquid is selected to be 6 ml/bottle.

The kit has the high sensitivity characteristic of a chemiluminescence immunoassay method, greatly reduces the cost, has low price compared with a foreign chemiluminescence detection system, does not need an expensive full-automatic chemiluminescence measuring instrument, and provides a new detection method for the detection of clinical Beta-2 microglobulin.

The kit provided by the invention adopts a fluorescein/fluorescein antibody system, and effectively overcomes the defects that Beta-2 microglobulin is not easy to coat and is easy to inactivate after coating, so that the difficulty of large-scale production of the kit is reduced, the sensitivity is high, the specificity is strong, the stability is good, the linear range is wide, the experiment cost is low, and the Beta-2 microglobulin can be more effectively detected.

Drawings

Statistical results of serum measurements of 1200 normal persons

FIG. 2 shows the correlation between the clinical fixed value specimen and the results of the comparison

Detailed Description

Example one preparation of the Beta-2 microglobulin chemiluminescence quantitative immunoassay kit of the present invention

The preparation method of this example includes the following steps:

1. preparing a solid-phase microporous plate: selecting high-purity and high-affinity fluorescein antibody, diluting to 2.5 mu g/ml by using 20mM carbonate buffer solution (pH9.6), coating at 4 ℃ overnight, spin-drying the liquid, adding sealing liquid into 250 mu l/hole, sealing at 37 ℃ for 2 hours, spin-drying the liquid, standing at room temperature for 24 hours to completely dry the luminescent plate, sealing and packaging by using an aluminum platinum bag, and finishing the preparation of the pre-coated luminescent plate coated by the fluorescein antibody.

2. Fluorescein labeling of capture antibody: after a high-purity high-affinity Beta-2 microglobulin capture antibody is selected and dialyzed for 4 hours at 4 ℃ by using 20mM phosphate buffer (pH7.4), a high-quality commercial fluorescein reagent is added according to the quantity of 30 mu g/mg, after reaction for 2 hours, the high-purity high-affinity Beta-2 microglobulin capture antibody is dialyzed for 2 hours at 4 ℃ by using 20mM phosphate buffer (pH7.4), transferred into a glass bottle, added with glycerol with the same volume and stored at-20 ℃ for later use.

3. Enzyme labeling of the detection antibody: selecting 2ml of high-purity high-affinity Beta-2 microglobulin detection antibody with the concentration of 2mg/ml and strictly matched with the selected capture antibody, dialyzing for 3 hours at 4 ℃ by using 2000ml of enzyme labeling buffer solution, adding 1mg of activated enzyme, reacting for 2 hours in a dark place, precipitating by using a saturated ammonium sulfate solution, centrifuging for 30 minutes at 4 ℃, redissolving the precipitate by using 10ml of phosphate buffer solution, adding isometric glycerol, and keeping at-20 ℃ for later use.

4. Mixing of labeled antibody liquid: before the kit is assembled, a capture antibody marked by fluorescein and a detection antibody marked by enzyme are respectively diluted by enzyme according to the proportion of 1: 2500 and 1: diluting at a ratio of 5000, mixing at equal volume, and packaging at a volume of 11 ml/bottle.

5. Preparation of luminescent substrate: based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value is 8.0-10.0, and the chemiluminescent substrate A solution is prepared and then subpackaged according to the amount of 6 mL/bottle;

based on 1000mL of the chemiluminescent substrate B solution, the chemiluminescent substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20, 51.58g of Na2HPO 4.12H 2O and 8.74g of NaH2PO 4.2H 2O, the pH value is 7.0-7.6, and the chemiluminescent substrate B solution is prepared and then subpackaged according to the amount of 6 mL/bottle.

6. Preparing Beta-2 microglobulin series standard products: commercial high-purity Beta-2 microglobulin is selected, the concentration of the microglobulin is determined by national standard substance contrast measurement, and calf serum is used for serial dilution: s6: 8.0. mu.g/ml, S5: 4.0. mu.g/ml, S4: 2.0. mu.g/ml, S3: 1.0. mu.g/ml, S2: 0.5. mu.g/ml, S1: 0.2. mu.g/ml, S0: 0 calf serum and 0.5 ml/bottle.

7. Preparation of concentrated washing solution: based on 1000mL of the concentrated washing solution, the concentrated washing solution comprises 4g of NaH2PO 4.2H2O, 58g of Na2HPO 4.12H2O, 175.32g of NaCl, 10mL of Tween-20 and 1mL of Proclin-300, the pH value of the concentrated washing solution is 7.0-7.6, and the concentrated washing solution is prepared and then is divided into two parts according to the requirement.

8. Each kit is assembled by one coating plate, one bottle of labeled antibody liquid, one bottle of luminescent substrate A liquid and one bottle of luminescent substrate B liquid, one bottle of concentrated washing liquid, one set of standard product, one part of instruction book and one sheet of sealing plate film.

Example two

In addition to the mixing of the labeled antibody fluid: before the kit is assembled, a capture antibody marked by fluorescein and a detection antibody marked by enzyme are respectively diluted by enzyme according to the proportion of 1: 2000 and 1: diluting at a ratio of 5000 and mixing in equal volume. The other steps are the same as those in the first embodiment.

EXAMPLE III

In addition to the mixing of the labeled antibody fluid: before the kit is assembled, a capture antibody marked by fluorescein and a detection antibody marked by enzyme are respectively diluted by enzyme according to the proportion of 1: 2000 and 1: 20000 and equal volume mixing after dilution. The other steps are the same as those in the first embodiment.

Example four

In addition to the mixing of the labeled antibody fluid: before the kit is assembled, a capture antibody marked by fluorescein and a detection antibody marked by enzyme are respectively diluted by enzyme according to the proportion of 1: 8000 and 1: diluting at a ratio of 5000 and mixing in equal volume. The other steps are the same as those in the first embodiment.

EXAMPLE five

In addition to the mixing of the labeled antibody fluid: before the kit is assembled, a capture antibody marked by fluorescein and a detection antibody marked by enzyme are respectively diluted by enzyme according to the proportion of 1: 8000 and 1: 20000 and equal volume mixing after dilution. The other steps are the same as those in the first embodiment.

EXAMPLE six

Method of use of the kit of the invention

1. Sample application

Taking out each component from the packaging box, balancing for 30 minutes at room temperature, tearing off the aluminum foil tape for packaging, placing the coating plate on a flat desktop, sequentially sucking 25 mul of each point of a standard product or each sample serum into each hole on the coating plate by using a micropipettor, adding the labeled antibody liquid according to the amount of 100 mul/hole, oscillating and mixing uniformly, and placing in a 37 ℃ water bath for reacting for 1 hour.

2. Washing machine

The coated plate was washed five times with 20mmol PBS, pH7.2, 0.05% Tween-20 buffer and then blotted dry.

3. Adding substrate

5ml of substrate A liquid and 5ml of substrate B liquid are taken, mixed evenly and immediately and rapidly added into each hole of the coated plate, and after shaking and mixing evenly, the mixture is reacted for 5 minutes at room temperature in a dark place.

4. Measuring

And (3) placing the coated plate in a chemiluminescence measuring instrument for measurement, reading, drawing a standard curve according to the concentration of the standard substance and the measured value, substituting the measured value of each sample into the standard curve, and calculating the concentration of each sample reagent.

EXAMPLE seven

Results of the actual cassette of the present invention for detecting clinical specimens

Clinical laboratory	Results of the experiment	Analysis of results
Clinical laboratory	Results of the experiment	Analysis of results	200 parts of normal human serum sample (actual measurement value shown in Table 1)	198 parts are all less than 3 mug/ml, and 2 parts are between 3 and 4 mug/ml. (see FIG. 1 for statistical chart)	The specificity is 99.00 percent
83 parts of clinical definite value specimen (actual measurement value in Table 2)	82 parts of the Chinese medicinal composition accord with clinical measurement values, and the coincidence rate is 98.8 percent	y is 0.9973x +1.3901r is 0.9955 (see fig. 2 for correlation diagram)			The specificity is 99.00 percent

Table 1: measurement results of 200 parts of normal human serum sample

Table 2: measurement results of 83 clinical fixed value specimens

Claims

1. A kit for quantitative detection of Beta-2 microglobulin in blood by chemiluminescence immunoassay is characterized by comprising:

1) pre-coated universal solid phase carriers;

2) a fluorescein-labeled capture antibody;

3) an enzyme-labeled detection antibody;

4) luminescent substrates required for the action of the above enzymes; and

5) beta-2 microglobulin series standard.

2. The kit of claim 1, wherein the pre-coat is a fluorescein antibody; the solid phase carrier is a microporous plate and a plastic pipe; the enzyme is horseradish peroxidase.

3. The kit of claim 1, wherein the luminophore for the chemiluminescent substrate is luminol, isoluminol.

4. The kit of claim 1, wherein the chemiluminescent substrate comprises solution A and solution B, wherein,

based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value of the chemiluminescent substrate A solution is 8.0-10.0;

based on 1000mL of the chemiluminescent substrate B solution, the chemiluminescent substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20, 51.58g of Na2HPO 4.12H 2O and 8.74g of NaH2PO 4.2H 2O, and the pH value of the chemiluminescent substrate B solution is 7.0-7.6.

5. A method for preparing the kit of claim 1, comprising the steps of:

1) preparing a universal solid phase carrier;

2) fluorescein labeling the capture antibody;

3) detecting an enzyme label of the antibody;

4) mixing and subpackaging a fluorescein labeled capture antibody and an enzyme labeled detection antibody;

5) preparing and subpackaging a luminescent substrate;

6) preparing Beta-2 microglobulin series standard substances and subpackaging;

7) and assembling to obtain the finished product kit.

6. The method of claim 5, wherein: in the step 1), the preparation method of the universal solid phase carrier comprises the following steps: selecting a high-purity and high-affinity fluorescein antibody, diluting the fluorescein antibody to a certain concentration by using a coating buffer solution, coating a solid phase carrier by a certain amount, sealing by using a sealing solution, airing, adding a drying agent, and then sealing in vacuum by using an aluminum foil bag.

7. The method of claim 5, wherein: in the step 2), the fluorescein labeling method of the capture antibody comprises the following steps: after a high-purity high-affinity Beta-2 microglobulin capture antibody is selected and dialyzed by using a fluorescein labeled buffer solution, a certain amount of high-quality commercial fluorescein reagent is added, after a certain time of reaction, the high-purity high-affinity Beta-2 microglobulin capture antibody is dialyzed by using the fluorescein labeled buffer solution again, and the high-purity high-affinity Beta-2 microglobulin capture antibody is stored at minus 20 ℃ for later use.

8. The method of claim 5, wherein: in the step 3), the enzyme labeling method for detecting the antibody comprises the following steps: selecting a Beta-2 microglobulin detection antibody which has high purity and high affinity and is strictly matched with the selected capture antibody, dialyzing by using an enzyme labeling buffer solution, adding a certain amount of activated enzyme, reacting for a certain time in a dark place, precipitating by using a saturated ammonium sulfate solution, centrifuging, redissolving the precipitate by using a phosphoric acid buffer solution, adding isometric glycerol, and keeping at-20 ℃ for later use.

9. The method of claim 5, wherein: in the step 4), the mixing and subpackaging method of the fluorescein labeled capture antibody and the enzyme labeled detection antibody comprises the following steps: before the kit is assembled, the capture antibody marked by fluorescein and the detection antibody marked by enzyme are diluted by enzyme diluent in proportion and then are subpackaged according to the required amount.

10. The method of claim 5, wherein: in the step 5), the preparation method of the luminescent substrate comprises the following steps: based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value is 8.0-10.0, and the chemiluminescent substrate A solution is prepared and then subpackaged according to the required amount; based on 1000mL of the chemiluminescent substrate B solution, the chemiluminescent substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20, 51.58g of Na2HPO 4.12H 2O and 8.74g of NaH2PO 4.2H 2O, the pH value is 7.0-7.6, and the chemiluminescent substrate B solution is prepared and then subpackaged according to required amount.

11. The method of claim 5, wherein: in the step 6), Beta-2 microglobulin series standard products are prepared: commercial high-purity Beta-2 microglobulin is selected, the concentration of the microglobulin is determined by national standard substance contrast measurement, and calf serum is used for serial dilution: s6: 8.0. mu.g/ml, S5: 4.0. mu.g/ml, S4: 2.0. mu.g/ml, S3: 1.0. mu.g/ml, S2: 0.5. mu.g/ml, S1: 0.2. mu.g/ml, S0: 0 calf serum.