CN114324855A - Detection kit for carbohydrate antigen CA72-4 - Google Patents

Abstract

The application relates to a detection kit for carbohydrate antigen CA 72-4. The kit comprises a first reagent, a second reagent, and optionally a calibrator. The first reagent comprises a buffer solution, a dispersing agent, a coagulant, a blocking agent, a surfactant and a preservative; the second reagent comprises buffer solution, latex microspheres coated by CA72-4 antibody, sucrose, casein and preservative. The kit has good accuracy, linearity, precision and stability.

Description

Detection kit for carbohydrate antigen CA72-4

Technical Field

The present disclosure relates to the fields of medicine, immunization, and in vitro diagnostics; in particular to a detection kit of sugar chain antigen 72-4(CA72-4) by an immune transmission turbidimetric method.

Background

CA72-4 is a carbohydrate antigen of mucin class recognized by cc49 and B72.3 monoclonal antibodies. CA72-4 has a molecular weight of 220kD to 400 kD. It is an IgG monoclonal antibody obtained in 1981 by Colcher et al immunizing mice with cancer cell membrane components of liver metastasis lesion of breast cancer, and can recognize TAG72 protein.

CA72-4 may be present in malignant tissues such as gastric cancer, colorectal cancer, breast cancer, etc., but is not expressed in non-epithelial malignant tumors and benign proliferative lesions. Therefore, the specificity of diagnosing malignant tumors is stronger. In recent years, CA72-4 has been clinically used for the diagnosis of gastric cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer and the like.

The current detection methodology of CA72-4 focuses on the chemiluminescent and enzyme-linked immunosorbent platforms. The detection method mainly comprises the following steps: electrochemiluminescence, magnetic particle chemiluminescence, enzyme-linked immunosorbent assay (DRG Instruments GmbH, germany).

For example, CN102288767A discloses a quantitative determination kit of CA72-4, which comprises CA72-4 magnetic separation reagent, enzyme reactant, reaction enhancer, and diluent.

CN104931703A provides a CA72-4 immunomagnetic bead test strip, which comprises a sample pad, a binding pad, a nitrocellulose membrane, a water absorption pad and a backing plate; the test paper is marked with anti-CA 72-4 monoclonal antibody CC49 and anti-CA 72-4 monoclonal antibody B72.3. After the test strip is used for immunochromatography, a magnetic strength detector is used for analyzing to obtain a quantitative result of CA 72-4.

CN105092852A provides a fluorescence immunoassay test strip for detecting CA72-4, the test strip comprises a sample pad, a quantum dot mark combining pad, a nitrocellulose membrane and absorbent paper; a quantum dot labeled anti-CA 72-4 monoclonal antibody CC49 coating is arranged on the quantum dot labeled bonding pad; the detection line is provided with an anti-CA 72-4 monoclonal antibody B72.3. After the test strip is used for immunochromatography, a fluorescence immunochromatography chip detector is used for analyzing to obtain a quantitative result of CA 72-4.

The chemiluminescence method has high sensitivity, strong specificity and good precision, but the reagent and the matched instrument have high price and small detection flux, and are not suitable for primary hospitals. With the implementation of the hierarchical medical policy, the patient flow of the primary hospital is increased, but the primary hospital is still sensitive to cost control.

Therefore, it is necessary to develop a high-throughput CA72-4 detection reagent that can be used in a quantitative manner in a general biochemical analyzer.

Disclosure of Invention

According to some embodiments, a kit for determining CA72-4 levels is provided, comprising a first reagent and a second reagent.

In the present application, CA72-4 refers to human CA 72-4. The term includes the various forms of molecules of CA72-4 throughout the cycle in vivo, such as, but not limited to, molecules produced by the CA72-4 gene during amplification, replication, transcription, splicing, processing, translation, modification, e.g., a pre-protein, a mature protein, a naturally occurring variant, a modified protein, or a fragment thereof.

By "determining the level of CA 72-4" is meant determining the amount of CA72-4 present, particularly the amount of protein levels, in a sample.

The terms "first," "second," and the like, in this application, are used solely to distinguish one product, step, feature, active ingredient from another, and do not denote a particular order, or degree.

In some specific embodiments, the first agent comprises or consists of:

glycine buffer 0.01M to 0.2M (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20M, or a range between any two values);

a dispersant 0.05M to 1M (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0M, or a range between any two values);

a polymerization promoter 0.1% w/v to 2% w/v (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0%, or a range between any two values);

a blocking agent in the range of 10mg/L to 200mg/L (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mg/L, or a range between any two values, which can be a decimal or integer);

0.05% w/v to 1.0% w/v (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0%, or a range between any two values) of surfactant;

a preservative 0.05% w/v to 2% w/v (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0%, or a range between any two values);

a pH of 5 to 8 (e.g., 5, 6, 7, 8, or a range between any two values, which may be a decimal or an integer).

In some specific embodiments, the second agent comprises or consists of:

hepes buffer 0.01M to 0.2M (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20M, or a range between any two values);

CA72-4 antibody package 0.1% w/v to 0.3% w/v (e.g., 0.1, 0.2, 0.3%),

Latex microspheres or a range between any two values);

sucrose 1% w/v to 10% w/v (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%, or a range between any two values, which may be a decimal or integer);

casein 0.2% w/v to 5% w/v (e.g., 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0%, or a range between any two values);

a preservative 0.05% w/v to 0.5% w/v (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5%, or a range between any two values);

a pH of 7 to 8 (e.g., a range between two values, which may be a decimal or an integer), preferably 7.5.

In some specific embodiments, the dispersant is selected from any one or combination of: sodium chloride, potassium chloride, magnesium sulfate and calcium chloride.

In some embodiments, the polymerization promoting agent is selected from any one or a combination of: polyvinylpyrrolidone, PEG12000, PEG8000, PEG 6000.

In some specific embodiments, the polymerization promoter is PVP 360000.

In some specific embodiments, the polyvinylpyrrolidone is 0.05%, 0.10%, 0.20%, 0.40%, 1.0%, 1.5%, 2.0% (w/v).

In some embodiments, the blocking agent is selected from any one or a combination of: HBR-5, HBR-6, HBR-7, HBR-21, HBR-22, HBR-23, HBR-24, HBR-28, HIER-R-001, and HIER-E-010D, CBS 10-KC.

In some specific embodiments, the blocking agent is HIER-E-010D.

In some embodiments, the surfactant is not: TW20, TW80, NP40, Thesit, Brij 23.

In some specific embodiments, the surfactant is distyrylphenol polyoxyethylene ether.

In some specific embodiments, the preservative is selected from any one or a combination of: sodium azide, PC300, sorbic acid and benzoic acid.

The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, fully human antibodies, humanized antibodies, and antibody fragments so long as they exhibit the desired antigen binding activity.

Antibodies (immunoglobulins) are tetrapeptide chain structures formed by two heavy chains and two light chains linked by interchain disulfide bonds. The constant regions of the heavy chains of immunoglobulins differ in their amino acid composition and arrangement, and thus are antigenically different. Accordingly, immunoglobulins can be classified as: IgM, IgD, IgG, IgA, and IgE.

The term "antigen-binding fragment" refers to fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. The antibody fragment retains the binding specificity of the parent antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2, Fd, dAb, nanobody, VHH domain, linear antibody, single chain antibody molecule (scFv).

The CA72-4 antibody is capable of recognizing and binding CA72-4 or an epitope thereof.

The CA72-4 antibody is derived from one or a combination of: mouse, rabbit, sheep, fowl, camel, recombinant expression.

The term "epitope" refers to the site on the CA72-4 antigen to which an immunoglobulin (or antibody) binds. Epitopes can be formed by adjacent amino acids, or by tertiary folding of proteins by non-adjacent amino acids. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope an antibody binds are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

In some specific embodiments, the first reagent does not comprise BSA.

In some specific embodiments, the second agent does not comprise BSA.

The present application has unexpectedly found that BSA can bind non-specifically to the detection system, thereby interfering with the detection results.

In some specific embodiments, the latex microspheres are polystyrene latex microspheres.

In some specific embodiments, the surface of the latex microspheres is modified with any one or a combination of groups selected from: carboxyl, amino, mercapto, hydroxyl.

In some specific embodiments, the CA72-4 antibody is covalently or non-covalently bound to the surface of the latex microsphere.

In some specific embodiments, the average particle size of the latex microspheres is 100nm to 350nm (e.g., 100, 150, 200, 250, 300, 350, or a range between any two of the foregoing values, which can be a decimal or integer).

It should be understood that the latex microspheres in the reagent are a mixture of a plurality of individual microspheres. For example, when referring to a particle size of 100nm, it is not intended that the particle size of each individual microsphere be 100nm whole, but rather that the size specification or model of the latex microspheres used, wherein the particle size range of the microspheres is around 100 nm.

In some embodiments, the reagents of the present application further comprise a calibrator and/or a quality control.

The calibrator is mainly used for calibrating a measurement system, evaluating a measurement program or assigning values to a sample to be measured. Thus, the calibrator contained a known concentration of CA72-4, and the value of the calibrator can be traced back to standards known in the art or commercially available.

One skilled in the art can prepare the calibrator at an appropriate concentration by himself or herself, and can use a commercially available calibrator, a product calibrator provided by a manufacturer, or the like, by using a method commonly used in the art.

In some embodiments, the calibrator comprises a known concentration of CA72-4, such as 0.0, 10.0, 20.0, 50.0, 100.0, 200.0U/mL.

The quality control product is prepared by adding a known amount of sample to be detected into a biological medium and is used for quality control. Those skilled in the art can prepare quality control materials with appropriate concentration by themselves by methods commonly used in the art, and can also use commercially available quality control materials and the like.

In some embodiments, the quality control comprises a known concentration of CA72-4, such as 8.0U/mL, 30U/mL.

In some embodiments, there is provided a method of detecting CA72-4 levels in a sample, comprising:

-providing a sample of the sample, and,

contacting the sample with a reagent of the present application,

-determining the level of CA72-4 in the sample.

The latex immunoturbidimetric detection technology is a homogeneous immunoassay developed and established on the basis of latex agglutination qualitative test, and can be used for accurately and quantitatively measuring various trace antigen substances and small molecule haptens. The principle is that according to the specific antibody coated sensitized latex particles, when meeting with corresponding CA72-4 antigen in a sample to be detected, agglutination reaction occurs to generate turbidity, the turbidity degree and the concentration of the detected object are in a functional relation through absorbance detection and comparison, and the content of the CA72-4 antigen in the sample to be detected can be measured through a generated dose-reaction curve.

Drawings

FIG. 1: the invention discloses a calibration curve of a CA72-4 latex immunoturbidimetric kit.

FIG. 2: the linear results of the CA72-4 latex immunoturbidimetry kit are disclosed.

FIG. 3: effect of blocking agent on sample measurement.

FIG. 4: effect of 0.2% TW80 surfactant on reagent stability.

FIG. 5: effect of 0.3% TW80 surfactant on reagent stability.

FIG. 6: effect of 0.3% TW20 surfactant on reagent stability.

FIG. 7: effect of 0.3% NP40 surfactant on reagent stability.

FIG. 8: effect of 0.3% Thesit surfactant on reagent stability.

FIG. 9: effect of 0.3% Brij23 surfactant on reagent stability.

FIG. 10: effect of 0.3% Emulgen a90 on reagent stability.

FIG. 11: the absorbance change was calibrated after acceleration.

Detailed Description

Example 1 preparation of the kits of the present application

1. Preparation of the first reagent:

the first reagent comprises:

the first reagent was prepared as described above at room temperature.

2. The second reagent is prepared as follows

0.5mL of a 200 nm-sized polystyrene latex solution (10% concentration) was added to 4.5mL of 0.05M MES (pH6.0) and 150. mu.l of EDC (carbodiimide, 5mg/mL) and allowed to stand at 37 ℃ for 1 hour;

the monoclonal antibody was diluted with 5ml of 0.05M Tris buffer (pH7.5) and immediately added to the above buffer to react at 37 ℃ for 2 hours;

1ml of 0.1M glycine buffer (pH7.0) was added thereto and stirred for 1 hour to terminate the reaction;

washing with 20ml of 100mM glycine buffer (pH7.0), centrifuging to remove supernatant, and washing 3 times;

resuspending the latex microspheres in a preservation buffer to disperse the latex microspheres into a white latex suspension;

the concentration of the polystyrene latex microspheres coated with the CA72-4 monoclonal antibody in the second reagent is 0.20%; the concentration of antibody in the second reagent corresponded to 0.065 g/L.

Composition of the preservation buffer:

the monoclonal antibodies in the second reagent are two different monoclonal antibodies (different epitopes are targeted, and antibodies that avoid steric hindrance may be selected so as not to interfere with each other and bind to the antigen). In one example, the mass ratio (or mass ratio) of the two mabs is 1: 1.

Optimization example 1 optimization of reagent accuracy and stability

1. Influence of blocking Agents

The kit was prepared as in example 1, except that different kinds of blockers were replaced and the effect of the measurement of the serum sample was observed. Different classes of blocking agents are as follows:

as can be seen in FIG. 3, 2 abnormally high sera recovered normal after the use of blockers 3 and 6 (both of which were HIER-E-010D, except for lot number). The blocking agent obviously improves the accuracy of the measured value of the reagent, but the stability of the reagent is weakened after the blocking agent is added.

In subsequent tests (see point 3 below in optimization example 1), the applicant unexpectedly found that the use of the blocking agent and the surfactant (distyrylphenol polyoxyethylene ether) simultaneously not only ensured the accuracy of the measurement of the kit, but also significantly improved the stability of the reagent, compared to the case of no surfactant.

2. Effect of the surfactant

The kit was prepared according to the method of example 1, except that different kinds and different concentrations of the surfactant were replaced.

(1) As can be seen from fig. 4 and table 1, the use of TW80 surfactant resulted in a relatively stable calibrated absorbance after the acceleration of the reagent, but the measured value of the sample decreased by an average of 10%, which affected the clinical use of the reagent (4 ℃ control, also referred to as 4 ℃ reagent in the figure).

TABLE 1.0.2% Effect of TW80 surfactant on measured values after acceleration of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	12.3	9.5	-22.8％
2	26.7	24.6	-7.9％
				3	45.9	41.3	-10.0％
4	38.9	34.9	-10.3％
				5	14.9	13.9	-6.7％
6	28.5	26.3	-7.7％

(2) As can be seen from fig. 5 and table 2, using 0.3% TW80 surfactant, the normalized absorbance did not change much after the reagent acceleration, but the sample measurements decreased by an average of 20%.

TABLE 2.0.3 Effect of TW80 surfactants on measured values after acceleration of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	8.6	6.9	-19.8％
2	11.2	8.8	-21.4％
				3	10.6	8.6	-18.9％
4	16.3	13.1	-19.6％
				5	30.7	27.4	-10.7％
6	34.8	28.6	-17.8％
				7	166.1	151.5	-8.8％

In summary, it can be seen that as the dosage of TW80 increases, the scaled absorbance is stable, but the decrease of the measured value of the sample increases.

(3) With TW20 surfactant, the accelerated reagent stability was particularly poor and both absorbance and sample measurements were significantly changed (fig. 6 and table 3).

TABLE 3.0.3% Effect of TW20 surfactant on measured values after acceleration of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	6.9	4.6	-33％
2	7.9	3.5	-56％
				3	47.1	42.1	-11％
4	54.5	55.3	1％
				5	97.6	84.9	-13％
6	56.6	53.8	-5％
				7	78.6	72.8	-7％

(4) Stability was acceptable using NP40 surfactant, but european union regulations did not allow the use of NP40 (fig. 7).

TABLE 4.0.3 Effect of NP40 surfactant on post-acceleration measurements of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	5.0	5.3	6％
2	8.6	8.4	-2％
				3	53.1	51.9	-2％
4	79.0	75.3	-5％
				5	110.0	106.3	-3％
6	71.2	67.5	-5％
				7	88.8	85.1	-4％

(5) The absorbance of the sample decreased significantly and unstably after the acceleration of the reagent using Thesit surfactant (fig. 8).

TABLE 5.0.3 Effect of% Thesit surfactant on measured values after acceleration of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	5.7	5.5	-4％
2	8.1	8.3	2.0％
				3	52.7	52.2	-1％
4	70.2	71.7	2％
				5	76.7	76.7	0％
6	67.6	67.5	0％
				7	86.9	86.8	0％

(6) The change of absorbance after the acceleration of the reagent is large and unstable by using Brij23 surfactant (FIG. 9).

TABLE 6.0.3 Effect of Brij23 surfactant on post-acceleration measurements of reagents

Sample(s)	Control at 4 deg.C	37-7 days	Deviation of
				1	5.1	5.3	4％
2	8.1	7.7	-5％
				3	52.1	52.2	0％
4	12.1	12.4	2％
				5	118.5	120.9	2％
6	57.0	56.5	-1％
				7	130.1	131.5	1％

According to tables 5 and 6, although the measured values of the samples containing Thesit and Brij23 did not change much before and after the reagents were accelerated, the calibration absorbances of the two groups after the acceleration were unstable. Therefore, calibration is required to be performed frequently, and the accuracy of the measured value of the sample after repeated calibration cannot be guaranteed. The performance of the reagent can be ensured only by using the reagent with stable sample measurement values before and after calibration and acceleration.

3. Combination of blocking agent and surfactant

Only when distyrylphenol polyoxyethylene ether is used as a surfactant, the absorbance and the measured value of the sample are stable after the acceleration of the reagent.

In conclusion, the accuracy and stability of the CA72-4 latex immunoturbidimetric assay kit can be optimized by using the blocking agent HIER-E-010D and distyrylphenol polyoxyethylene ether (Emulgen A90) in combination (Table 7 and FIG. 10).

TABLE 7.0.3 influence of distyrylphenol polyoxyethylene ether on the measured value after acceleration of the reagent

Optimization example 2. repetitive optimization procedure

A kit was prepared according to the method of example 1, except that: different kinds of polymerization promoters. It was found that the use of PVP360000 in the first reagent significantly improved the reproducibility of the measurements.

TABLE 8 Effect of Using different polymerization promoters on repeatability

Test example 1 Linear Range of the kits of the present application

A kit was prepared according to the method of example 1, and high-value serum of 200U/mL was tested, and the linearity of the kit was examined according to the method of isocratic dilution with purified water, and the results are as follows.

TABLE 9 kit linearity

Dilution ratio	Measured value 1	Measured value 2	Mean value	Theoretical value	AbsoluteDeviation of	Relative deviation of
							0	0.0	0.0	0.00	0.42	-0.42	-100.0％
0.0125	2.4	2.5	2.46	2.93	-0.47	-15.9％
							0.025	5.0	5.1	5.03	5.44	-0.41	-7.6％
0.05	10.0	10.1	10.04	10.46	-0.42	-4.0％
							0.1	20.4	20.4	20.36	20.51	-0.15	-0.7％
0.2	39.9	39.8	39.83	40.61	-0.78	-1.9％
							0.3	62.8	62.7	62.76	60.70	2.06	3.4％
0.4	79.7	78.6	79.16	80.80	-1.64	-2.0％
							0.5	105.2	104.5	104.88	100.89	3.99	4.0％
0.6	119.6	119.1	119.36	120.99	-1.63	-1.3％
							0.7	143.5	144.5	144.03	141.08	2.95	2.1％
0.8	158.9	159.4	159.16	161.18	-2.02	-1.3％
							0.9	182.2	183.4	182.81	181.27	1.54	0.8％
1	199.3	198.2	198.76	201.37	-2.60	-1.3％

As can be seen from Table 9, the kit can reach a linear detection range of 200U/mL, and the concentration of the clinical conventional sample CA72-4 is mostly concentrated below 200U/mL, which meets the clinical application (FIG. 2).

Test example 2 reproducibility

The kit was prepared according to the method of example 1, 3 samples of different concentrations were tested, each for 10 consecutive tests, and the reproducibility of the test (expressed in precision) was calculated;

TABLE 10 measurement of precision

Serial number	Sample	1	Sample 2	Sample 3
					1	2.7	5.4	14.8
2	2.6	5.3	15.1
				3	2.6	5.2	15.3
4	2.6	5.1	14.9
				5	2.6	5.2	15.3
6	2.4	5.2	15.2
				7	2.6	5.5	15.1
8	2.5	5.3	15.0
				9	2.4	5.4	15.2
10	2.6	5.1	15.7
				Mean value	2.55	5.26	15.16
Variance (variance)	0.10	0.13	0.25
				CV	3.9％	2.5％	1.65％

As can be seen from Table 10, the kit has good detection repeatability, the reference range of CA72-4 is 10U/mL, and the kit has good repeatability in the reference range, so that the kit meets the clinical application.

Test example 3 reagent stability

A kit is prepared according to the method of example 1, a part of the reagent is put into an incubator at 37 ℃ for 7 days in an accelerated manner, then the reagent is taken out and is calibrated by being compared with the reagent at 4 ℃, a sample is detected, and the accelerated stability of the reagent is observed.

TABLE 11 measured value change of sample after acceleration

Sample(s)	Control at 4 deg.C	7 days at 37 DEG C	Variations in
				Sample 1	4.2	4.4	5％
Sample
	2	7.8	7.5	-4％
Sample
	3	47.5	48.7	3％
Sample
	4	61.5	63.2	3％
Sample
	5	95.5	97.9	3％
Sample
	6	60.1	60.8	1％
Sample
	7	79.6	80.4	1％

As can be seen from the data in FIG. 11 and Table 11, the change rate of the measured value of the calibration and sample is controlled within 5% after the reagent is accelerated for 7 days, and the reagent is relatively stable and meets the clinical requirement.

Claims (9)

1. A kit for determining CA72-4 levels, comprising:

a first reagent, and

a second reagent;

wherein the first reagent comprises:

the second reagent comprises:

the dispersant is selected from any one or combination of: sodium chloride, potassium chloride, magnesium chloride, calcium chloride, magnesium sulfate;

the polymerization promoter is selected from any one or combination of the following: polyvinylpyrrolidone, PEG12000, PEG8000, PEG 6000; preferably polyvinylpyrrolidone;

the preservative is selected from any one or a combination of: sodium azide, PC300, sorbic acid, benzoic acid; sodium azide is preferred.

2. The kit of claim 1, the first reagent does not comprise BSA for determining CA72-4 levels.

3. The kit for determining the level of CA72-4 according to claim 1 or 2, wherein the blocking agent is HIER-E-010D.

4. The kit for determining the level of CA72-4 according to any one of claims 1 to 3, wherein the surfactant is distyrylphenol polyoxyethylene ether.

5. The kit for determining the level of CA72-4 according to any one of claims 1 to 4, wherein the polymerization promoter is PVP 360000.

6. The kit of any one of claims 1 to 5 for determining the level of CA72-4, the CA72-4 antibody being derived from one or a combination of: mouse, rabbit, sheep, fowl, camel, and artificial recombination.

7. The kit for determining the level of CA72-4 according to any one of claims 1 to 6, wherein:

the latex microspheres are polystyrene latex microspheres;

the surface of the latex microsphere is modified with any one or a combination of the following groups: carboxyl, amino, mercapto, hydroxyl;

the average particle size of the latex microspheres is 100nm to 350 nm.

8. The kit for determining the level of CA72-4 according to any one of claims 1 to 7, further comprising a calibrator and/or a quality control.

9. The kit for determining CA72-4 levels according to any one of claims 1 to 8, the surfactant being not selected from any one of: TW20, TW80, NP40, Thesit, Brij 23.

Images