CN111781372A

CN111781372A - Alpha 1-AT immunoturbidimetry detection kit based on mixed antibody and preparation and use methods thereof

Info

Publication number: CN111781372A
Application number: CN202010610053.0A
Authority: CN
Inventors: 芮双印; 高耀辉
Original assignee: Anhui Daqian Bio Engineering Ltd
Current assignee: Anhui Daqian Bio Engineering Ltd
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2020-10-16

Abstract

The invention provides a α 1-AT immunoturbidimetry detection kit based on a mixed antibody, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises HEPES, NaOH and EDTA-Na₂PEG-6000, PEG-8000 and the like; the reagent R2 comprises NaH₂PO₄·2H₂O、Na₂HPO₄·12H₂O、NaCl、EDTA‑Na₂The invention also provides a preparation and use method of the detection kit, wherein the anti- α 1-AT mixed antibody is prepared by mixing the anti- α 1-AT monoclonal antibody I, the anti- α 1-AT monoclonal antibody II and the anti- α 1-AT polyclonal antibody III, and the like, and the BSA and the anti- α 1-AT mixed antibody are adopted to obtain the anti- α 1-AT mixed antibody by mixing the high-specificity monoclonal antibody with the high-sensitivity polyclonal antibody, so that the quality of the detection kit is improvedThe specificity and the sensitivity of the kit are improved.

Description

Alpha 1-AT immunoturbidimetry detection kit based on mixed antibody and preparation and use methods thereof

Technical Field

The invention relates to the field of genetic engineering and immunological determination and analysis, in particular to an alpha 1-AT immunoturbidimetry detection kit based on a mixed antibody and a preparation and use method thereof.

Background

Alpha 1 antitrypsin (alpha 1-AT) is a glycoprotein, contains 10% -20% of sugars, is mainly synthesized by the liver, and is widely distributed in normal human serum and body fluids. Alpha 1 antitrypsin is the most important protease inhibitor in serum, and has inhibitory effect on other enzymes such as thrombin and urokinase. Alpha 1 antitrypsin is also an acute phase reaction protein, and in inflammatory diseases, the alpha 1 antitrypsin can enter tissue fluid through capillaries, is often very high in concentration at local inflammation and has a certain limiting effect on acute inflammatory diseases.

AT present, the methods for detecting alpha 1-AT mainly comprise two methods, namely antigen-antibody reaction and enzyme rate method. The enzyme rate method has insufficient detection sensitivity, large deviation of clinical detection, poor repeatability and stability and difficult clinical popularization and application. In the nineties of the twentieth century, immunoturbidimetry was used as a homogeneous assay for the routine study of α 1-AT assays, which can be automated in biochemical instruments for clinical testing.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope. The preparation of the monoclonal antibody is that the hybridoma cell is obtained by using B lymphocyte treated by specific antigen and myeloma cell through cell fusion method, positive clone strain is obtained after HAT culture medium screening and ELISA titer detection, finally cell culture is carried out to obtain the monoclonal antibody after the cell is injected into the abdominal cavity of an animal for abdominal water culture, and supernatant fluid/ascites is collected and purified.

Stimulation of the body by multiple epitopes correspondingly produces a wide variety of monoclonal antibodies, which when mixed together are polyclonal. The polyclonal antibody is prepared by directly injecting antigen into animal body for immunization, performing immunization for 3-4 times, collecting blood, centrifuging to obtain supernatant, and purifying to obtain the polyclonal antibody after ELISA detection.

The existing alpha 1-AT immunoturbidimetric assay kit applied in clinic can be divided into two categories, namely a polyclonal antibody kit and a monoclonal antibody kit, from the aspect of antibody use. Practical application finds that the polyclonal antibody reagent has the problem of poor detection specificity, and the monoclonal antibody reagent has the problem of poor sensitivity. Among them, the monoclonal antibody has high specificity, can specifically recognize a specific epitope, but has low sensitivity for antigen detection. The polyclonal antibody can be exactly complementary with the monoclonal antibody, the high sensitivity can make up the low sensitivity problem of the monoclonal antibody, but the problem of poor detection specificity exists.

Accordingly, there is a need for a mixed antibody-based α 1-AT immunoturbidimetry assay kit employing both a high-specificity monoclonal antibody and a high-sensitivity polyclonal antibody, and a method for preparing and using the same, thereby improving the specificity and sensitivity of the assay for the kit sample.

Disclosure of Invention

The invention aims to provide a mixed antibody-based alpha 1-AT immunoturbidimetry detection kit simultaneously adopting a high-specificity monoclonal antibody and a high-sensitivity polyclonal antibody, and a preparation and use method thereof.

The invention adopts the following technical scheme to solve the technical problems:

a mixed antibody-based alpha 1-AT immunoturbidimetry detection kit comprises a reagent R1 and a reagent R2 which are independent of each other, and a double-liquid component;

the reagent R1 comprises the following components in parts by weight: HEPES 20-30g/L, NaOH 0.2-0.8g/L, NaCl5-10g/L, EDTA-Na₂1-1.2g/L, PEG-600010-20 g/L, PEG-80005-10 g/L, Tween 200.1-0.6%, NaN₃0.01g/L, and the solvent is purified water;

the reagent R2 comprises the following components in parts by weight: NaH₂PO₄·2H₂O 1-2g/L，Na₂HPO₄·12H₂O 6-10g/L，NaCl 5-10g/L，EDTA-Na₂1-1.2g/L BSA 1-5g/L anti- α 1-AT hybrid antibody 1-20%, NaN₃0.01g/L, and the solvent is purified water;

wherein the anti-alpha 1-AT mixed antibody is formed by mixing an anti-alpha 1-AT monoclonal antibody I, an anti-alpha 1-AT monoclonal antibody II and an anti-alpha 1-AT polyclonal antibody III.

In a preferred embodiment of the present invention, the anti- α 1-AT mixed antibody contains 0.5-2mg/mL of anti- α 1-AT monoclonal antibody I, 0.1-1mg/mL of anti- α 1-AT monoclonal antibody II, and 0.1-1mg/mL of anti- α 1-AT polyclonal antibody III.

As one of the preferable modes of the invention, the reagent R1 comprises the following components in percentage by weight: HEPES23.83g/L, NaOH 0.4g/L, NaCl9.0g/L, EDTA-Na₂1.1g/L, PEG-600015 g/L, PEG-80008 g/L, Tween 200.5%, NaN₃0.01g/L, and the solvent is purified water.

As one of the preferable modes of the invention, the reagent R2 comprises the following components in percentage by weight: NaH₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O 9.71g/L，NaCl 9.0g/L，EDTA-Na₂1.1g/L, BSA2.4g/L, 10% of anti- α 1-AT mixed antibody and NaN₃0.01g/L, and the solvent is purified water.

As one of the preferable modes of the invention, the kit also comprises α 1-AT calibrator, wherein the α 1-AT calibrator comprises the components with the corresponding content of NaH₂PO₄·2H₂O 1-2g/L，Na₂HPO₄·12H₂O6-10g/L, BSA 5-10g/L, trehalose 0.4-4g/L, α 1-AT protein 0.1-20g/L, NaN₃0.01g/L, 5-15% of glycerol and purified water as a solvent.

As one of the preferable modes of the invention, the α 1-AT calibration product comprises the following components and the corresponding content of NaH₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O9.71g/L, BSA8g/L, trehalose 2g/L, α 1-AT protein 10g/L, NaN₃0.01g/L, 10% of glycerol and the solvent is purified water.

In a preferred embodiment of the present invention, the anti- α 1-AT monoclonal antibody I, the anti- α 1-AT monoclonal antibody II, the anti- α 1-AT polyclonal antibody III, and the α 1-AT protein in the α 1-AT calibrator are all conventional products that are commercially available in the art. The anti- α 1-AT monoclonal antibody I and the anti- α 1-AT monoclonal antibody II may be monoclonal antibodies capable of specifically binding to α 1-AT. The anti-alpha 1-AT polyclonal antibody III is preferably a rabbit anti-alpha 1-AT polyclonal antibody.

The preparation method of the alpha 1-AT immunoturbidimetry detection kit based on the mixed antibody comprises the following steps:

(1) preparation of anti-alpha 1-AT Mixed antibody

Preparing two monoclonal antibodies of anti-alpha 1-AT and one polyclonal antibody of anti-alpha 1-AT, namely an anti-alpha 1-AT monoclonal antibody I, an anti-alpha 1-AT monoclonal antibody II and an anti-alpha 1-AT polyclonal antibody III;

mixing the anti-alpha 1-AT monoclonal antibody I, the anti-alpha 1-AT monoclonal antibody II and the anti-alpha 1-AT polyclonal antibody III to prepare an anti-alpha 1-AT mixed antibody, wherein the content of the anti-alpha 1-AT monoclonal antibody I, the content of the anti-alpha 1-AT monoclonal antibody II and the content of the anti-alpha 1-AT polyclonal antibody III in the anti-alpha 1-AT mixed antibody are respectively 0.5-2mg/mL, 0.1-1mg/mL and 0.1-1 mg/mL;

(2) preparation of alpha 1-AT immunoturbidimetry kit calibrator

Preparation of a reagent R1:

according to the component content of the reagent R1, mixing the components in the same container, and uniformly mixing to obtain a reagent R1;

preparing a reagent R2:

mixing the anti-alpha 1-AT mixed antibody prepared in the step (1) and the rest other component substances in the same container according to the component content of the reagent R2, and uniformly mixing to prepare a reagent R2;

preparing an alpha 1-AT calibrator:

α 1 the corresponding component and corresponding content of the calibration product are NaH₂PO₄·2H₂O 1-2g/L，Na₂HPO₄·12H₂O6-10g/L, BSA 5-10g/L, trehalose 0.4-4g/L, α 1-AT protein 0.1-20g/L, NaN₃0.01g/L, 5-15% of glycerol and purified water as a solvent;

and (3) mixing the components in the same container according to the component content of the alpha 1-AT calibrator, and uniformly mixing to obtain the alpha 1-AT calibrator.

A use method of the alpha 1-AT immunoturbidimetry detection kit based on the mixed antibody comprises the following specific steps:

(1) sucking 3 μ L of sample, adding 240 μ L of reagent R1, and incubating at 37 deg.C for 3-5 min;

(2) then adding 60 mu L of reagent R2 for mixing and fully reacting;

(3) reading a light absorption value A1 after 1min, reading a light absorption value A2 after 5min, and calculating delta A;

(4) the calibration method is 6-point calibration, a full-automatic biochemical analyzer is adopted for detection, and the concentrations of calibrators are respectively set as follows: 0g/L, 0.18g/L, 0.37g/L, 0.74g/L, 1.47g/L and 2.94 g/L; and (4) according to the calibration value, calculating the content of alpha 1-AT in the sample according to the delta A.

Compared with the prior art, the invention has the advantages that:

(1) compared with other methods, the method utilizes an immunoturbidimetry method to measure the alpha 1-AT, and adopts a high-specificity monoclonal antibody to mix with a high-sensitivity polyclonal antibody to prepare the anti-alpha 1-AT mixed antibody, so that the specificity and the sensitivity of sample detection are improved;

(2) according to the invention, the reagent R1 adopts PEG-6000 and PEG-8000 to be mixed for use, so that the absorbance value of the reagent reaction is improved, and the sensitivity of the reagent detection is improved;

(3) according to the invention, a certain amount of glycerol and trehalose are added into the calibration solution, so that the stability of the calibration solution is ensured, and the detection accuracy of the kit is improved.

Drawings

FIG. 1 is a graph showing the linear relationship between the kit of the present invention and a commercial α 1-AT detection kit in example 6;

FIG. 2 is a linear range linear regression plot of the kit of the invention in example 6.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

The mixed antibody-based alpha 1-AT immunoturbidimetry detection kit comprises a reagent R1 and a reagent R2 which are independent of each other, and a double liquid component.

The reagent R1 comprises the following components in parts by weight: HEPES 20g/L, NaOH 0.2g/L, NaCl 5g/L, EDTA-Na₂1g/L, PEG-600010 g/L, PEG-80005 g/L, Tween 200.1%, NaN₃0.01g/L, and the solvent is purified water.

The reagent R2 comprises the following components in parts by weight: NaH₂PO₄·2H₂O 1g/L，Na₂HPO₄·12H₂O 6g/L，NaCl 5g/L，EDTA-Na₂1g/L of BSA, 1g/L of anti- α 1-AT hybrid antibody, 1% of NaN₃0.01g/L and the solvent is purified water, wherein the anti- α 1-AT mixed antibody is formed by mixing an anti- α 1-AT monoclonal antibody I, an anti- α 1-AT monoclonal antibody II and a rabbit anti- α 1-AT polyclonal antibody III.

Furthermore, in the anti-alpha 1-AT mixed antibody, the content of the anti-alpha 1-AT monoclonal antibody I is 0.5mg/mL, the content of the anti-alpha 1-AT monoclonal antibody II is 0.1mg/mL, and the concentration of the rabbit anti-alpha 1-AT polyclonal antibody III is 0.1 mg/mL.

In addition, the α 1-AT immunoturbidimetry detection kit based on the mixed antibody also comprises α 1-AT calibrator, wherein the α 1-AT calibrator comprises the components with the corresponding content of NaH₂PO₄·2H₂O 1g/L，Na₂HPO₄·12H₂O6 g/L, BSA 5g/L, trehalose 0.4g/L, α 1-AT protein 0.1g/L, NaN₃0.01g/L, 5% of glycerol, and the solvent is purified water.

Example 2

The reagent R1 comprises the following components in parts by weight: HEPES 30g/L, NaOH 0.8g/L, NaCl 10g/L, EDTA-Na₂1.2g/L, PEG-600020 g/L, PEG-800010 g/L, Tween 200.6%, NaN₃0.01g/L, and the solvent is purified water.

The reagent R2 comprises the following components in parts by weight: NaH₂PO₄·2H₂O 2g/L，Na₂HPO₄·12H₂O 10g/L，NaCl 10g/L，EDTA-Na₂1.2g/L, BSA 5g/L, anti- α 1-AT MIXED ANTIBODY 20%, NaN₃0.01g/L and the solvent is purified water, wherein the anti- α 1-AT mixed antibody is formed by mixing an anti- α 1-AT monoclonal antibody I, an anti- α 1-AT monoclonal antibody II and a rabbit anti- α 1-AT polyclonal antibody III.

Furthermore, in the anti-alpha 1-AT mixed antibody, the content of the anti-alpha 1-AT monoclonal antibody I is 2mg/mL, the content of the anti-alpha 1-AT monoclonal antibody II is 1mg/mL, and the concentration of the rabbit anti-alpha 1-AT polyclonal antibody III is 1 mg/mL.

In addition, the α 1-AT immunoturbidimetry detection kit based on the mixed antibody also comprises α 1-AT calibrator, wherein the α 1-AT calibrator comprises the components with the corresponding content of NaH₂PO₄·2H₂O 2g/L，Na₂HPO₄·12H₂O10 g/L, BSA10g/L, trehalose 4g/L, α 1-AT protein 20g/L, NaN₃0.01g/L, 15% of glycerol and the solvent is purified water.

Example 3

The reagent R1 comprises the following components in parts by weight: HEPES23.83g/L, NaOH 0.4g/L, NaCl9.0g/L, EDTA-Na₂1.1g/L, PEG-600015 g/L, PEG-80008 g/L, Tween 200.5%, NaN₃0.01g/L, and the solvent is purified water.

The reagent R2 comprises the following components in parts by weight: NaH₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O9.71g/L，NaCl 9.0g/L，EDTA-Na₂1.1g/L, BSA2.4g/L, 10% of anti- α 1-AT mixed antibody and NaN₃0.01g/L and the solvent is purified water, wherein the anti- α 1-AT mixed antibody is formed by mixing an anti- α 1-AT monoclonal antibody I, an anti- α 1-AT monoclonal antibody II and a rabbit anti- α 1-AT polyclonal antibody III.

Furthermore, in the anti-alpha 1-AT mixed antibody, the content of the anti-alpha 1-AT monoclonal antibody I is 1.5mg/mL, the content of the anti-alpha 1-AT monoclonal antibody II is 0.5mg/mL, and the concentration of the rabbit anti-alpha 1-AT polyclonal antibody III is 0.5 mg/mL.

In addition, the α 1-AT immunoturbidimetry detection kit based on the mixed antibody also comprises α 1-AT calibrator, wherein the α 1-AT calibrator comprises the components with the corresponding content of NaH₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O9.71g/L, BSA8g/L, trehalose 2g/L, α 1-AT protein 10g/L, NaN₃0.01g/L, 10% of glycerol and the solvent is purified water.

Example 4

The preparation method of the mixed antibody based α 1-AT immunoturbidimetry assay kit of examples 1-3 includes the following steps:

(1) preparation of anti-alpha 1-AT Mixed antibody

mixing the anti-alpha 1-AT monoclonal antibody I, the anti-alpha 1-AT monoclonal antibody II and the anti-alpha 1-AT polyclonal antibody III to prepare an anti-alpha 1-AT mixed antibody, wherein the contents of the anti-alpha 1-AT monoclonal antibody I, the anti-alpha 1-AT monoclonal antibody II and the anti-alpha 1-AT polyclonal antibody III in the anti-alpha 1-AT mixed antibody respectively correspond to the above examples;

(2) preparation of alpha 1-AT immunoturbidimetry kit calibrator

Preparation of a reagent R1:

preparing a reagent R2:

preparing an alpha 1-AT calibrator:

Example 5

This example describes the method of using the mixed antibody based α 1-AT immunoturbidimetry assay kit of examples 1-3 above.

The analysis method comprises the following steps: a two-point end-point method;

the reaction direction is as follows: raising reaction;

the calibration method comprises the following steps: Lomit-Log (4P) or SPLINE processing;

measuring wavelength: 340 nm;

measuring temperature: 37 ℃;

sample preparation: reagent R1: reagent R2 ═ 3: 240: 60(μ L)

The testing steps are as follows: pipetting 3 μ L of sample, adding 240 μ L of reagent R1, incubating at 37 deg.C for 3-5min, adding 60 μ L of reagent R2, reading absorbance A1 after 1min, reading absorbance A2 after 5min, and calculating Δ A.

The calibration method comprises the following steps: 6 point calibration, adopting Hitachi 7180 full-automatic biochemical analyzer (or other brands and models), detecting, setting the concentration of the calibrator as follows: 0. 0.18, 0.37, 0.74, 1.47 and 2.94 g/L.

And calculating the content of alpha 1-AT in the sample according to the calibration value and the delta A.

Example 6

This example was used to evaluate the mixed antibody based α 1-AT immunoturbidimetry assay kit of the above examples:

(1) and (3) verifying linear correlation:

the reagent prepared by the formula of the example 3 is compared with α 1-AT detection kits of some marketed companies approved by the State food and drug administration for detection, 100 clinical serum samples are detected, the detection results are shown in the following table 1, a correlation curve of the kit and α 1-AT detection reagents sold by some marketed companies is obtained (see figure 1; in figure 1, the abscissa represents the detection result value of α 1-AT detection reagents sold by some marketed companies in unit g/L; the ordinate represents the detection result value of the kit in unit g/L), and the detection results show that the linear correlation curve of the two kits is y 1.0087-0.0653X, and the correlation curve is y 1.0087-0.0653XNumber R²The greater correlation between the two is illustrated at 0.9897.

TABLE 1 comparison of the Linear correlation between the kit of the present invention and the alpha 1-AT detection kit commercially available from some commercial companies

(2) Linear range verification

The recombinant α 1-AT and physiological saline are used for preparing test products with the concentration of 4.0g/L, 2.0g/L, 1.0g/L, 0.5g/L and 0g/L (physiological saline control), the concentration of each test product is measured by using the kit of the invention, a linear regression equation is obtained by using the dilution concentration as an independent variable and the measurement result as a dependent variable (in the figure 2; in the figure 2, the abscissa represents the dilution concentration and the unit g/L; the ordinate represents the measurement concentration and the unit g/L), the relative deviation of the measurement result is calculated, the detection and calculation results are shown in the table 2, and the results show that the linear regression equation between the measurement result and the dilution concentration is y 0.9575+0.0318X, and the correlation coefficient R is R²The linear relation is good when the value is 0.9999, and the linear range can reach 4 g/L.

TABLE 2 validation of the Linear Range of the kit of the invention

(3) Accuracy verification

And taking one portion of traceable high-value serum quality control and one portion of traceable low-value serum quality control, detecting for 10 times by using the kit, taking a mean value, and comparing with a quality control target value. The results show that the detection values have smaller relative deviation and higher accuracy than the target values, which are shown in Table 3.

Accuracy verification results of the kit described in Table 3

(4) Precision verification

Taking high value and low value of clinical serum samples detected by the kit on sale, continuously detecting the same serum sample for 10 times by using the kit, and calculating the coefficient of variation of the kit. The precision detection data are shown in the following table 4, and the detection results show that the kit has smaller variation coefficients of 0.34% and 1.73% and better precision when detecting high-value and low-value samples.

Precision verification results of the kit shown in Table 4

(5) Verification of sensitivity and specificity

50 parts of positive serum and 50 parts of negative serum are detected and screened by adopting an imported indirect ELISA method alpha 1-AT detection kit, a commercially available turbidimetric immunoassay kit prepared by using an anti-alpha 1-AT polyclonal antibody, a commercially available turbidimetric immunoassay kit prepared by using an anti-monoclonal antibody and the 100 parts of serum sample are synchronously detected by adopting the kit, the kit is set to be positive when being higher than a reference standard and negative when being lower than the reference standard according to the determination standard of each kit, the sensitivity and specificity of each kit are calculated by taking the imported indirect ELISA method detection kit as a gold standard, and the result is shown in a table 5. The result shows that the kit has higher sensitivity and specificity compared with two kits sold in the market. The invention has the outstanding advantages that the kit has higher sensitivity compared with a monoclonal antibody preparation kit and has higher specificity compared with a kit prepared by more cloned antibodies, thereby greatly improving the accuracy of clinical detection and meeting the requirement of clinical detection.

Table 5 comparison of sensitivity and specificity of the kit with those of the commercially available kit

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 and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A mixed antibody-based alpha 1-AT immunoturbidimetry detection kit is characterized by comprising reagent R1 and reagent R2 which are independent of each other, as well as double liquid components;

2. The mixed antibody-based alpha 1-AT immunoturbidimetry assay kit according to claim 1, wherein the anti-alpha 1-AT mixed antibody contains 0.5-2mg/mL of anti-alpha 1-AT monoclonal antibody I, 0.1-1mg/mL of anti-alpha 1-AT monoclonal antibody II, and 0.1-1mg/mL of anti-alpha 1-AT polyclonal antibody III.

3. The α 1-AT immunoturbidimetry assay kit based on the mixed antibody of claim 1, wherein the reagent R1 comprises HEPES23.83g/L, NaOH 0.4g/L, NaCl9.0g/L, EDTA-Na₂1.1g/L, PEG-600015 g/L, PEG-80008 g/L, Tween 200.5%, NaN₃0.01g/L, and the solvent is purified water.

4. The α 1-AT immunoturbidimetry assay kit based on the mixed antibody of claim 1, wherein the reagent R2 comprises NaH as a component and in a content corresponding thereto₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O9.71g/L，NaCl 9.0g/L，EDTA-Na₂1.1g/L, BSA2.4g/L, anti- α 1-AT hybrid antibody 10%, NaN₃0.01g/L, and the solvent is purified water.

5. The α 1-AT immunoturbidimetry assay kit based on the mixed antibody of any one of claims 1-4, further comprising a α 1-AT calibrator, wherein the α 1-AT calibrator comprises the components and corresponding contents of NaH₂PO₄·2H₂O 1-2g/L，Na₂HPO₄·12H₂O6-10g/L, BSA 5-10g/L, trehalose 0.4-4g/L, α 1-AT protein 0.1-20g/L, NaN₃0.01g/L, 5-15% of glycerol and purified water as a solvent.

6. The α 1-AT immunoturbidimetry assay kit of claim 5, wherein the α 1-AT calibrator comprises NaH as a component and in an amount corresponding thereto₂PO₄·2H₂O 1.48g/L，Na₂HPO₄·12H₂O9.71g/L, BSA8g/L, trehalose 2g/L, α 1-AT protein 10g/L, NaN₃0.01g/L, 10% of glycerol and the solvent is purified water.

7. A method for preparing a mixed antibody based α 1-AT immunoturbidimetry assay kit according to any of claims 1-6, comprising the steps of:

(1) preparation of anti-alpha 1-AT Mixed antibody

(2) preparation of alpha 1-AT immunoturbidimetry kit calibrator

Preparation of a reagent R1:

preparing a reagent R2:

preparing an alpha 1-AT calibrator:

8. The method for using the mixed antibody based alpha 1-AT immunoturbidimetry assay kit according to any of claims 1-6, comprising the following specific steps:

(2) then adding 60 mu L of reagent R2 for mixing and fully reacting;