CN111060689B

CN111060689B - Endometrial cancer detection kit

Info

Publication number: CN111060689B
Application number: CN201911061439.4A
Authority: CN
Inventors: 周标; 陈艳华; 席云
Original assignee: Guangzhou Bokang Biotechnology Co ltd
Current assignee: Guangdong Jucheng Biotechnology Co.,Ltd.
Priority date: 2019-11-01
Filing date: 2019-11-01
Publication date: 2020-12-25
Anticipated expiration: 2039-11-01
Also published as: CN111060689A

Abstract

The invention relates to the technical field of endometrial cancer diagnosis and detection reagents, in particular to a kit for detecting the concentrations of galectin-3 and CA 125. The kit comprises a tracer labeled CA125 antibody, a tracer labeled galectin-3 antibody, a CA125 antibody coated plate and a galectin-3 antibody coated plate. The kit provided by the invention has high sensitivity and good stability, and can also be applied to endometrial cancer diagnosis and detection kits for disease monitoring and curative effect evaluation.

Description

Endometrial cancer detection kit

Technical Field

The invention relates to the technical field of endometrial cancer diagnosis and detection reagents, and in particular relates to an endometrial cancer detection kit.

Background

Endometrial cancer is a group of epithelial malignancies that occur in the endometrium, often in perimenopausal and postmenopausal women, and is the third most common gynecological malignancy that leads to death. With the change of living habits, the incidence rate of endometrial cancer is gradually increased. Currently, the etiology of endometrial cancer is not clear, and it is difficult to prevent the endometrial cancer, so that early detection and treatment of the endometrial cancer are needed. Typically, clinical diagnosis of endometrial cancer requires reliance on diagnostic curettage, but this approach often has certain risks and complications.

The tumor marker is a substance which is characterized to exist in malignant tumor cells, is abnormally produced by the malignant tumor cells, or is produced by the stimulation response of a host to the tumor, and can reflect the occurrence and the development of the tumor and monitor the response of the tumor to treatment. Among them, galectin-3 is related to proliferation, apoptosis, adhesion and the like of regulatory cells, the expression level of the galectin-3 is closely related to the malignancy of tumors, and related researches show that the over-expression of the galectin-3 is related to abnormal hyperplasia and malignant change of endometrium, and is also closely indistinguishable from differentiation, metastasis, invasion and the like of endometrial cancer, so that the galectin-3 is a valuable index for early diagnosis and prognosis judgment of clinical endometrial cancer. CA125 is a glycoprotein that can be bound by the monoclonal antibody OC125, and is commonly found in the serum of patients with epithelial ovarian tumors, and is absent from normal human ovaries. Generally, CA125 is more sensitive but less specific at the time of diagnosis. In addition, the value is not high in the early stage of nearly half of patients, so that the single-stage diagnosis of the ovarian epithelial cancer cannot be realized.

Chinese patent CN02828218.3 discloses a method and kit for the pre-diagnosis of cancer, the method comprising determining the concentration of galectin-3 in a blood sample by reacting the blood sample with a monoclonal antibody of galectin-3; comparing the determined concentration of galectin-3 with the concentration of galectin-3 in a blood sample of a normal human; if the measured concentration is higher than the galectin-3 concentration in the blood of a normal human, the prognosis is of a risk of contracting cancer. The method is simple and easy, but only can preliminarily predict the occurrence of the galectin-3 in the precancerous stage and the early stage so as to predict whether the precancerous stage and the early stage have the risk of infecting the cancer, and the specific cancer cannot be qualitatively judged.

In order to overcome the defects of single marker detection, the domestic application has already adopted the report of joint detection of a plurality of markers, for example, Chinese patent CN201410162945.3 discloses a kit for quickly diagnosing early and middle stages of ovarian cancer and a detection method thereof, the kit comprises monoclonal antibodies respectively prepared from galectin-3, transthyretin, cancer antigen-125 and human epididymis protein 4, can comprehensively detect ovarian cancer, better judge the benign and malignant degree of pelvic lump before or after menopause, better perform differential diagnosis on gynecological benign ovarian lump and cyst and ovarian cancer, has the characteristics of high sensitivity and high accuracy, and the sensitivity and the accuracy of the kit can reach more than 96 percent. However, the kit and the detection method have problems of large amount of required serum, long analysis time, and the like due to a large number of detection items.

However, the components involved in the kit are complex, so that the requirement on storage conditions is high, the operation of the kit is complex, and how to simplify the components of the kit under the condition of ensuring the detection result is a problem to be solved urgently at present.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention provides a kit for detecting endometrial cancer. The galectin-3 combined with CA125 is adopted to screen early-stage endometrial cancer, so that the occurrence of the endometrial cancer is prevented, and meanwhile, the kit can also be applied to disease monitoring and curative effect evaluation of the endometrial cancer.

In order to achieve the above object, the present invention provides the following technical solutions:

an endometrial cancer detection kit comprises a tracer marked CA125 antibody, a tracer marked galectin-3 antibody, a CA125 antibody coated plate and a galectin-3 antibody coated plate.

Preferably, the kit also comprises a calibrator solution, a reaction buffer solution, an enhancement solution and/or a concentrated washing solution which are prepared into a series of standard solutions by adopting CA125 and galectin-3 antigen.

Preferably, the tracer labeled CA125 antibody is prepared from lanthanide and CA125 monoclonal antibody in a mass ratio of 1: 3-5;

the tracer labeled galectin-3 antibody is prepared from lanthanide and galectin-3 monoclonal antibody in a mass ratio of 1: 3-5.

In one embodiment, the tracer-labeled CA125 antibody is produced by EU³⁺-N₂- [ P-Isocyanato-benzyl group]The-sodium diethylenetriamine tetracetate and the CA125 monoclonal antibody are prepared according to the mass ratio of 1: 3. What is needed is

The tracer labeled galectin-3 antibody is expressed by EU³⁺-N₂- [ P-Isocyanato-benzyl group]The-sodium diethylenetriamine tetracetate and the CA125 monoclonal antibody are prepared according to the mass ratio of 1: 3.

Preferably, the lanthanide is Eu³⁺。

Preferably, the reaction buffer is a solution containing bovine serum albumin with a mass concentration of 0.1-10%.

Further preferably, the mass concentration of bovine serum albumin in the reaction buffer is 0.1-5%, and even more preferably, the mass concentration of bovine serum albumin in the reaction buffer is 0.2-0.8%.

As an implementation mode, the reaction buffer solution comprises the components and the concentration of 1-10g/L NaCl, 0.1-10 thousandth EDTA, 0.1-20 thousandth FPC, 0.1-10 thousandth mouse serum, 0.1-10 thousandth thiomersal, 0.1-10% pyrus communis, pH7.0-8.0 and 5-80mmol/L Tris-HCl, wherein the concentration of the FPC and the mouse serum is in a volume ratio, and the concentration of the EDTA, the thiomersal and the pyrus communis is in a mass ratio.

Preferably, the enhancement solution is a solution containing Triton-X-100, trioctylphosphine oxide, beta-benzoyltrifluoroacetone, acetic acid and potassium hydrogen phthalate.

As an embodiment, the enhancement solution is a solution containing 0.1-5.5% by volume of Triton-X-100, 0.1-2.2g/L of trioctylphosphine oxide, 0.005-0.01g/L of beta-benzoyltrifluoroacetone, 1-30% by volume of glacial acetic acid and 0.005-0.01 g/L.

Preferably, the concentrated wash solution contains a surfactant at a concentration of 2-10% by volume, and a buffer at a pH of 7.0-8.0.

Further preferably, the surfactant is one or more of tween 20, tween 40, tween 60 and tween 80.

Preferably, the CA125 antigen series concentration range in the calibrator solution is 10-1000 ng/mL; the concentration range of the galectin-3 antigen series in the calibrator solution is 10-1000 ng/mL.

The antigen is prepared by the following method: the galectin-3 and cancer antigen-125 (CA-125) genes are cloned to a eukaryotic expression vector by a molecular cloning method, the expression of protein is realized in mammalian cells, and the antigen is obtained after purification and can also be used as a standard substance; the antigen can also be prepared using conventional methods.

The antibody is obtained by immunizing mammals with the antigen prepared by the method to obtain the corresponding monoclonal antibody. The mammal is a mouse or rabbit, preferably a rabbit.

The coating in the CA125 antibody-coated plate and the galectin-3 antibody-coated plate refers to the treatment comprising the following steps:

(1) pretreating the monoclonal antibody of galectin-3 or CA125, and coating the holes of the microplate with the pretreated monoclonal antibody by using a buffer solution A;

(2) cover the microplate and incubate;

(3) discarding the coating solution, washing the microplate, adding the buffer solution B, washing twice, removing the washing solution, and drying in the air;

(4) and (3) sealing by using a phosphate buffer sealing solution with the pH value of 7.0-7.5 for later use.

Preferably, the pretreatment in the step (1) refers to the constant temperature treatment of the monoclonal antibody mixture of galectin-3 and CA125 in two stages, wherein the temperature of the first stage is-3 to-1 ℃, the temperature of the second stage is 0 to 4 ℃, and the treatment time of the two stages is 10 to 30 min.

Preferably, the buffer a in step (1) is a carbonate or bicarbonate buffer (pH 9.6);

the buffer solution B in the step (2) is 3-8% of sucrose-PBST;

preferably, the incubation temperature in the step (2) is 2-6 ℃, and the incubation time is 16-24 h;

as a preferred embodiment, the monoclonal antibodies of galectin-3 and CA125 in step (1) are diluted with buffer A to a concentration of 1-4. mu.g/mL each, and buffer B is added in an amount of 100. mu.L per well in step (3).

The invention also provides a preparation method of the endometrial cancer detection kit, which comprises the following steps:

preparation of S1 antibody coated plate

Preparation of S1.1 monoclonal antibody

Immunizing a mouse with the antigen obtained by the method to obtain a corresponding monoclonal antibody;

s1.2 monoclonal antibody coating

(1) Mixing monoclonal antibodies of galectin-3 and CA125 and pretreating, and coating the holes of the microplate with the pretreated monoclonal antibodies by using a buffer solution A;

(2) cover the microplate and incubate;

(4) sealing with phosphate buffer solution with pH value of 7.0-7.5;

preparation of S2 tracer-labeled antibody

The tracer-labeled CA125 antibody is produced by EU³⁺-N₂- [ P-Isocyanato-benzyl group]-diethylenetriamineMixing sodium tetraacetate and the CA125 monoclonal antibody in a mass ratio of 1:3-5 to prepare the monoclonal antibody;

the tracer labeled galectin-3 antibody is expressed by EU³⁺-N₂- [ P-Isocyanato-benzyl group]The-diethylenetriamine tetra-sodium acetate and the galectin-3 monoclonal antibody are mixed according to the mass ratio of 1: 3-5.

Preferably, when the kit further comprises a calibrator solution, a reaction buffer solution, an enhancing solution and/or a concentrated washing solution which are prepared from CA125 and galectin-3 antigen and are used as a series of standard solutions, the method further comprises the following steps:

preparation of S3 calibrator

Respectively taking CA125 and galectin-3 antigen standard substances as references, and diluting the CA125 antigen with a buffer solution into 6 liquid standard substances with the concentration of 10-1000 ng/mL;

diluting galectin-3 antigen with buffer solution into 6 liquid standard substances with concentration of 10-1000 ng/mL;

preparation of S4 reaction buffer

Adding bovine serum albumin with the mass ratio of 6% into a solution containing 1-10g/L NaCl, 0.1-10% of EDTA, 0.1-20% of FPC, 0.1-10% of mouse serum, 0.1-10% of merthiolate, 0.1-10% of pyrrhodin, pH7.0-8.0, 5-80mmol/L Tris-HCl (the concentration of the FPC and the mouse serum is volume ratio, and the concentration of the EDTA, the merthiolate and the pyrrhodin is mass ratio);

preparation of S5 enhancing solution

Preparing aqueous solution containing 0.1-5.5% by volume of Triton-X-100, 0.1-2.2g/L of trioctylphosphine oxide, 0.005-0.01g/L of beta-benzoyl trifluoroacetone, 1-30 per thousand by volume of glacial acetic acid and 0.005-0.01g/L of potassium hydrogen phthalate to obtain enhancement solution;

s6 test buffer solution, enhancing solution, concentrated washing solution and Eu prepared in the above way³⁺Subpackaging the labeled antibody and the calibrator, and packaging the prepared coated plate of the CA125 antibody and the galectin-3 antibody by using an aluminum foil bag, wherein the calibrator is frozen and dried after being subpackaged; and assembling the subpackaged and packaged articles to obtain the kit.

Compared with the prior art, the invention has the following beneficial effects:

the endometrial cancer detection kit provided by the invention adopts combined detection of galectin-3 and CA125, and has high sensitivity and strong specificity; and the stability is good, and the low-temperature storage time can reach 18 months. The kit can be used for preparing a diagnostic detection kit for endometrial cancer.

Detailed Description

The kit for rapid diagnosis of ovarian cancer in early and intermediate stages according to the present invention will be described in detail with reference to the following specific examples. Unless otherwise stated, the reagents and instruments involved in the following examples of the present invention are all commercially available products, and the products of different manufacturers do not bring obvious differences in technical effects. Wherein Eu³⁺-N₂- [ P-Isocyanato-benzyl group]Sodium-diethylenetriaminetetracetate was purchased from PE company, usa.

The serum to be tested was provided by the third subsidiary hospital of Zhongshan university.

Cross-linked Sepharose 6B column from GE, USA.

Time-resolved fluoroimmunoassay analyzer, PE corporation, usa, model Auto DELFIA 1235.

Preparation of galectin-3 and CA125 antigens: the galectin-3 and cancer antigen-125 (CA-125) genes are cloned to a eukaryotic expression vector by a molecular cloning method, the expression of protein is realized in mammalian cells, and the antigen is obtained after purification and can also be used as a standard substance; the antigen can also be prepared using conventional methods.

Example 1

The embodiment provides a kit for diagnosing and detecting endometrial cancer, which comprises a tracer labeled CA125 antibody, a tracer labeled galectin-3 antibody, a CA125 antibody coated plate, a galectin-3 antibody coated plate, a calibrator solution for preparing a series of standard solutions by adopting CA125 and galectin-3 antigens, a reaction buffer solution, an enhancement solution and a concentrated washing solution; the preparation method of the kit comprises the following steps:

preparation of S1 antibody coated plate

Preparation of S1.1 monoclonal antibody

s1.2 monoclonal antibody coating

(1) The monoclonal antibodies of galectin-3 and CA125 were mixed and pretreated, and used

Diluting the pretreated monoclonal antibody to 1ug/mL by using a carbonate buffer solution A with the pH of 9.6, and then coating the diluted monoclonal antibody on a culture plate;

(2) cover the microplate and incubate;

(3) discard coating solution, wash microplate, and add buffer B5% sucrose-PBST buffer

Washing twice with the solution, removing the washing solution, and drying in the air;

(4) sealing by using a phosphate buffer sealing solution with the pH value of 7.5 for later use;

the pretreatment in the step (1) is to perform two-stage constant temperature treatment on the monoclonal antibody mixture of galectin-3 and CA125, wherein the temperature of the first stage is-3 to-1 ℃, the temperature of the second stage is 0 to 4 ℃, and the treatment time of the two stages is 15 min.

Preparation of S2 tracer-labeled antibody:

the tracer-labeled CA125 antibody is produced by EU³⁺-N₂- [ P-Isocyanato-benzyl group]Mixing sodium diethylenetriamine tetracetate and the CA125 monoclonal antibody according to the mass ratio of 1:3, and magnetically stirring for 12 hours at 30 ℃. The next day, the mixture is subjected to cross-linked agarose gel 6B column chromatography, eluent is collected according to the flow rate of 1 ml/tube, the Eu fluorescence intensity of each tube is measured, the first Eu elution peak is collected, and the obtained Eu-CA125 liquid calibration substance is subjected to vacuum freeze drying to obtain the freeze-dried powder calibration substance.

The tracer labeled galectin-3 antibody is expressed by EU³⁺-N₂- [ P-Isocyanato-benzyl group]Mixing-sodium diethylenetriamine tetracetate and the galectin-3 monoclonal antibody according to the mass ratio of 1:3, and magnetically stirring for 12 hours at 30 ℃. The next day, the mixture was subjected to column chromatography on cross-linked sepharose 6BCollecting eluent according to the flow rate of 1 ml/tube, measuring the Eu fluorescence intensity of each tube, collecting the first Eu elution peak, and carrying out vacuum freeze drying on the obtained Eu-galectin-3 liquid calibrator to obtain the freeze-dried powder calibrator.

Preparation of S3 calibrator

Respectively taking CA125 and galectin-3 antigen standard substances as references, and diluting the CA125 antigen with a buffer solution into liquid standard substances with the concentrations of 10ng/mL, 20ng/mL, 50ng/mL, 200ng/mL, 400ng/mL and 1000 ng/mL;

diluting galectin-3 antigen with buffer solution into liquid standard substance with concentration of 10ng/mL, 20ng/mL, 50ng/mL, 200ng/mL, 400ng/mL, 1000 ng/mL;

the buffer solution is 8g/L BSA, 20g/L NaCl and 4g/L NaN₃pH of 1mL/L Thimerosal

7.5, 8.3mmol/L Tris-HCl solution.

Preparation of S4 reaction buffer

Bovine serum albumin with the mass ratio of 6 percent is added into a solution containing 10g/L NaCl, 3 thousandth EDTA, 10 thousandth FPC, 8 thousandth mouse serum, 10 thousandth thiomersal, 10 percent pyruvre, pH7.8, 55mmol/L Tris-HCl (the concentration of the FPC and the mouse serum is volume ratio, and the concentration of the EDTA, the thiomersal and the pyruvre is mass ratio).

Preparation of S5 enhancing solution

Triton-X-100 with the volume concentration of 4.5 percent, trioctylphosphine oxide with the volume concentration of 2.2g/L, beta-benzoyl trifluoroacetone with the volume concentration of 0.008g/L, glacial acetic acid with the volume concentration of 30 per thousand and potassium hydrogen phthalate solution with the volume concentration of 0.01g/L are prepared into water solution, namely the enhancement solution.

For the experiment buffer solution, the enhancement solution, the concentrated washing solution and the Eu prepared above³⁺And (3) subpackaging the labeled antibody and the calibrator, and packaging the prepared coated plate of the CA125 antibody and the galectin-3 antibody by using an aluminum foil bag, wherein the label is subpackaged at a rate of 500 mu L/bottle or the calibrator is freeze-dried at a rate of 500 mu L/bottle. And assembling the subpackaged and packaged articles to obtain the kit.

S5.1 preparation of concentrated washing solution

50mmol/L Tris-containing solution at pH7.8 of 1g/L Triton X-100 with 22.5g/L NaCl

4mL/L of Tween-20 with the volume concentration of 8% is added into the HCl solution, and the mixture is mixed uniformly.

S6 test buffer solution, enhancing solution, concentrated washing solution and Eu prepared in the above way³⁺And (3) subpackaging the labeled antibody and the calibrator, and packaging the prepared coated plate of the CA125 antibody and the galectin-3 antibody by using an aluminum foil bag, wherein the label is subpackaged at a rate of 500 mu L/bottle or the calibrator is freeze-dried at a rate of 500 mu L/bottle. And assembling the subpackaged and packaged articles to obtain the kit.

Experimental example 1

The kit of embodiment 1 is used for simultaneously detecting the contents of endometrial tumor markers CA125 and galectin-3, and comprises the following steps:

sample preparation: the serum sample is venous blood collected by a serum tube, and the supernatant is obtained by centrifugation.

The plate washing liquid is prepared by uniformly mixing concentrated washing liquid and distilled water in a ratio of 1: 25.

Each plate of the reaction plate is provided with 2 holes of the calibrator (A-F), and 50 mu L of calibrator and the volume to be calibrated are added into the corresponding holes in sequence

The sample is tested, 50 mu L of reaction buffer solution reacts for 1 hour at 25 ℃, and washing solution is used for washing for 4 times; then adding 100 mu L of reagent containing lanthanide-labeled CA125 antibody and lanthanide-labeled galectin-3 antibody into each well, reacting for 1 hour at 25 ℃, washing for 6 times by using a plate washing solution, adding 100 mu L of enhancement solution into each well, oscillating for 5 minutes, detecting a fluorescence signal value by using a time-resolved fluorescence immunoassay instrument, respectively obtaining fluorescence values of the calibrator solution and a sample to be detected, drawing a standard curve according to the concentration of the calibrator solution and the fluorescence value corresponding to the calibrator solution with the concentration, and fitting the standard curve by taking the fluorescence signal value as a vertical coordinate Y and the concentration of the corresponding CA125 antigen or galectin-3 antigen in the calibrator solution as a horizontal coordinate X; the sample concentration is then found on this standard curve by interpolation.

The performance of the kit provided by the present invention is specifically illustrated as follows

1.1 sensitivity test

220 samples are respectively detected by using the kit for detecting endometrial tumor markers CA125 and galectin-3 and a common enzyme-free diagnosis kit, wherein (218 samples of CA125 and galectin-3 (all purchased from Shanghai enzyme-linked biotechnology limited) and 2 blank control samples) are detected by using the two detection kits, and if the initial detection results of the samples are inconsistent, the two detection kits are used for detecting the samples again.

And (3) displaying a detection result: with the detection result of the enzyme-linked immunosorbent assay (ELISA) method as a reference, the detection results of 1 sample are inconsistent for the CA125 sample. The sensitivity of the time-resolved immunofluorescence method reaches 99.5%.

TABLE 1

	Positive for	Negative of
			Enzyme-linked immunosorbent assay (ELISA) method	215	5
Example 2 kits	217	3

TABLE 2

	Positive for	Negative of
			Enzyme-linked immunosorbent assay (ELISA) method	215	5
Example 2 kits	216	4

The results of 2 samples were inconsistent with the galectin-3 sample. The sensitivity of the time-resolved immunofluorescence method reaches 99.1% by taking the detection result of the enzyme-linked immunosorbent assay as reference.

Experimental example 2: stability analysis of the kit

The detection and diagnosis kit provided in example 1 of the present invention was stored at high temperature (37 ℃) and low temperature (4 ℃), 3 low/medium/high samples of CA125 and galectin-3 at different concentrations were measured every 2 days, the test was stopped when at least one of the deviation of the detection results of CA125 and galectin-3 from the initial detection values was 10% or more, the storage time was recorded, and the results are shown in table 3. As can be seen from the test data in Table 3, the kit provided by the invention has excellent stability and can be stored at low temperature for more than 12 months.

Table 3 stability test data of the kit provided in example 2 of the present invention

As can be seen from the data in Table 3, the kit provided in example 2 of the present invention can be stored at low temperature for up to 18 months, and has good stability.

The same detection method is adopted to detect the common enzyme-free diagnosis kit (same as the experimental example 1), and the result shows that the preservation time of the kit provided by the invention is obviously longer than that of the common enzyme-free diagnosis kit, namely the kit provided by the invention has better storage stability.

Experimental example 3

Sample preparation: the serum sample was venous blood collected by a serum tube, and the supernatant was collected by centrifugation.

100 persons in early stage of endometrial cancer and 30 persons in non-endometrial cancer patients are randomly selected, the kit for diagnosing and detecting endometrial cancer provided by the embodiment 1 of the invention is used for detecting the content of tumor markers CA125 and galectin-3 according to the method of the experimental example 1, the obtained detection results are compared with the content of CA125 and galectin-3 in the endometrial cancer patients and the non-endometrial cancer patients respectively, and the detection results are counted as shown in the following table 4.

Wherein, the range of CA125 in normal human serum is 0-35IU/mL, and the value of galectin-3 is 0.

CA125 in the serum of the patient with endometrial cancer ranges from more than or equal to 40 IU/mL; the galectin-3 content is 5IU/mL or more.

TABLE 4 clinical test results

As can be seen from the clinical data of the table above, the kit provided by the invention has higher goodness of fit with the actual result in clinical examination.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for detecting endometrial cancer is characterized by comprising the following components: tracer marking CA125 antibody, tracer marking galectin-3 antibody, CA125 antibody coated plate and galectin-3 antibody coated plate; preparing a calibrator solution, a reaction buffer solution, an enhancement solution and a concentrated washing solution of a series of standard solutions by adopting CA125 and galectin-3 antigen; the enhancement solution is a solution containing Triton-X-100, trioctylphosphine oxide, beta-benzoyl trifluoroacetone, acetic acid and potassium hydrogen phthalate;

the reaction buffer solution is a solution containing bovine serum albumin with the mass concentration of 0.1-10%;

(1) pretreating monoclonal antibodies of galectin-3 and CA125, and then coating the holes of the microplate with the pretreated monoclonal antibodies by using a buffer solution A; the buffer A is a carbonate or bicarbonate buffer with pH of 9.6;

(2) cover the microplate and incubate;

(3) discarding the coating solution, washing the microplate, adding the buffer solution B, washing twice, removing the buffer solution B, and drying in the air; the buffer solution B is 3-8% of sucrose-PBST;

(4) sealing with phosphate buffer solution with pH value of 7.0-7.5; the pretreatment in the step (1) is to perform two-stage constant temperature treatment on the monoclonal antibody mixture of galectin-3 and CA125, wherein the temperature of the first stage is-3 to-1 ℃, the temperature of the second stage is 0 to 4 ℃, and the treatment time of the two stages is 10 to 30 min;

the concentration range of the CA125 antigen series in the calibrator solution is 10-1000 ng/mL; the concentration range of the galectin-3 antigen series in the calibrator solution is 10-1000 ng/mL;

the tracer labeled CA125 antibody is prepared from lanthanide and a CA125 monoclonal antibody in a mass ratio of 1: 3-5;

the tracer labeled galectin-3 antibody is prepared from lanthanide and galectin-3 monoclonal antibody in a mass ratio of 1: 3-5;

the lanthanide is Eu³⁺。