CN111089975A

CN111089975A - Chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody and preparation method thereof

Info

Publication number: CN111089975A
Application number: CN201911366039.4A
Authority: CN
Inventors: 高智玲; 翟涛; 高威; 孙成艳; 何浩会
Original assignee: Dirui Medical Technology Co Ltd
Current assignee: Dirui Medical Technology Co Ltd; Changchun Dirui Industrial Co Ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-05-01

Abstract

The invention provides a chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody and a preparation method thereof, belonging to the technical field of immunoassay. The kit comprises: 1) a calibrator; 2) coating the TOXO antigen with magnetic beads; 3) a luminescent marker labeled anti-human IgG antibody; 4) item diluent 5) chemiluminescent excitation liquids a and B and a cleaning liquid. The invention also provides a preparation method of the chemiluminescence immunoassay determination kit for detecting the Toxoplasma gondii IgG antibody. The kit has the advantages of rapid detection, wide linear range, high sensitivity, less interference factors, low cost and the like.

Description

Chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody and a preparation method thereof.

Background

Toxoplasmosis is a widespread infectious disease caused by intracellular protozoan parasites (called toxoplasma gondii). Such diseases can infect humans and other warm-blooded animals. The transmission route may be by feeding food contaminated with oocysts, direct contact with poultry, or via placental infection, Toxoplasma transmission by blood transfusion or organ transplantation. In the normal adult population, toxoplasmosis is generally a benign course with the clinical manifestations mostly being asymptomatic or mildly symptomatic (headache, sore throat, weakness); very few cases are accompanied by lymphadenitis. Severe body disorders such as myocarditis, hepatitis, pneumonia, meningoencephalitis and retinochoroiditis occur in very rare cases.

The prevalence of positive serum tests gradually increased with age, suggesting a subsequent infection. Cell-mediated immune responses are generally involved in the control of parasitic infections. It is therefore more common for patients on immunosuppressive therapy (to prevent organ rejection or as an anti-tumor therapy) to develop a symptomatic course. Toxoplasma encephalitis has been demonstrated to be one of the major causes of death in patients with acquired immunodeficiency syndrome.

If a pregnant woman develops toxoplasma infection, the fetus can be compromised, such as by spontaneous abortion, premature birth or dead fetus, because the pathogen can spread to the fetus through the placenta. If Toxoplasma gondii is infected in the first trimester of pregnancy, severe damage to the central nervous system of the fetus can be caused, and the fetus can finally die. If Toxoplasma gondii is infected during the third month of pregnancy, hydrocephalus, mental retardation and psychomotor depression, blindness and cerebral calcification can result. Toxoplasma gondii infections are most common in the third trimester of pregnancy, however, and can lead to retinal choroiditis and other eye damage. Injury to the central nervous system and latent asymptomatic infection can ultimately lead to disease development.

The level of specific Toxoplasma gondii IgG antibody is gradually increased and reaches the peak 2-5 months after the clinical symptoms appear. Thus, the presence of IgG antibodies will help identify patients who have become infected. This is particularly important for the selection of appropriate precautions for women of the reproductive age in question.

The currently commonly used methods for detecting Toxoplasma gondii IgG antibodies include Radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA), but the two methods have many defects, such as radioactive pollution of RIA, short half-life of a marker, radioactive damage to an operator, complex operation, long detection time and the like; ELISA has the defects of low sensitivity, narrow detection range, long detection time, poor repeatability and the like.

Disclosure of Invention

The invention aims to overcome the defects of short effective period, complex operation, low sensitivity and high detection cost of a reagent in the prior art, and provides a chemiluminescence immunoassay kit for detecting Toxoplasma gondii IgG antibody and a preparation method thereof.

The invention firstly provides a chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody, which comprises:

1) a calibrator; 2) coating the TOXO antigen with magnetic beads; 3) a luminescent marker labeled anti-human IgG antibody; 4) Item diluent 5) chemiluminescent excitation liquids a and B and a cleaning liquid.

Preferably, in the TOXO antigen coated by the magnetic beads, the particle size of the magnetic beads is 0.05-3.0 μm, and the concentration of the TOXO antigen coated by the magnetic beads is 0.5-5%.

Preferably, the anti-human IgG labeled with the chemiluminescent label is mouse anti-human IgG or goat anti-human IgG.

Preferably, the labeling molar ratio of the anti-human IgG antibody to the chemiluminescent label is 1: 3-20.

Preferably, the chemiluminescent label is an acridinium ester, an acridinium ester derivative, and ruthenium terpyridyl.

Preferably, the item diluent is a buffer solution with pH of 6.0-8.0 and containing salt, surfactant, preservative and protein.

Preferably, the chemiluminescence excitation liquid A is a solution of hydrogen peroxide and nitric acid, and the chemiluminescence excitation liquid B is a solution of sodium hydroxide; the cleaning solution is PB buffer solution.

Preferably, the calibrator is formulated with an antibody to TOXO IgG.

The invention also provides a preparation method of the chemiluminescence immunoassay determination kit for detecting the Toxoplasma gondii IgG antibody, which comprises the following steps:

preparation of calibrator

Diluting TOXO IgG antibody into calibrator with PBS buffer containing calf serum, and subpackaging into 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, 200U/mL;

second, magnetic bead coated TOXO antigen solution preparation

Fully mixing the magnetic beads by using a vortex mixer, adding the suspended liquid into a centrifugal tube, placing the centrifugal tube on a magnetic separation frame for 1-3 min, removing supernatant, adding a sealing agent, fully mixing by using the vortex mixer, then placing the centrifugal tube on the magnetic separation frame, removing supernatant, repeating the sealing step for 3 times, adding the sealing agent again, fully mixing by using the vortex mixer, adding a coated antibody, fully mixing by vortex, incubating for 2-4 h at room temperature by using a 360-degree rotary mixer, placing the centrifugal tube on the magnetic separation frame, removing supernatant, adding a cleaning buffer, fully mixing by using the vortex mixer, placing the centrifugal tube on the magnetic separation frame, removing supernatant, repeatedly cleaning, resuspending the magnetic beads by using a storage buffer, collecting and storing;

thirdly, preparation of anti-human IgG antibody solution marked by luminescent marker

Putting the antibody into a centrifuge tube, adding a carbonic acid buffer solution, fully and uniformly mixing, adding a luminescent marker solution after uniformly mixing, centrifuging at room temperature by using a centrifuge, sealing the centrifuge tube by using a sealing film, then putting the centrifuge tube into a light-proof cassette, then putting the cassette into a gas bath constant-temperature oscillator, uniformly mixing for 2-4 h, adding a lysine sealing solution, putting the gas bath constant-temperature oscillator into the light-proof cassette, uniformly mixing at medium speed for 0.5-2 h, purifying the sealed antibody on a sephadex G250 column, eluting by using a PB buffer solution, collecting step by step, and storing the collected antibody solution at 2-8 ℃;

four, project diluent

The dilution of the item is a buffer solution containing salt, surfactant, preservative and protein, and has a pH of 6.0-8.0.

The invention has the advantages of

1. Acridinium ester and the like are selected as chemiluminescent labeling materials, the materials have direct chemiluminescence as energy transition generated when an excited state returns to a ground state, enzyme participation is not needed, time and cost are saved, and the system linearity range is wide.

2. The detection kit is a chemiluminescence immunoassay, and the method has the advantages of simple operation, no radioactivity risk and short detection time, and can detect target substances such as antigens and antibodies; on the other hand, the toxoplasma IgG chemiluminescence immunoassay kit and the chemiluminescence immunoassay instrument form a closed system, thereby reducing the error of manual operation and improving the sensitivity and accuracy of the whole system.

Drawings

FIG. 1 is a standard curve of relative luminescence values detected by a test calibrator using the inventive TOXO IgG antibody chemiluminescence quantitative kit, wherein the abscissa is concentration, the unit is U/mL, and the ordinate is luminescence value.

Detailed Description

In order to make the advantages, purposes and methods of the present invention more comprehensible, embodiments accompanying figures and specific examples are described in detail below. In the following description, numerous details are set forth to provide an understanding, but the present invention may be practiced otherwise than as described.

According to the invention, in the TOXO antigen coated by the magnetic beads, the particle size of the magnetic beads is 0.05-3.0 μm, and the concentration of the TOXO antigen coated by the magnetic beads is 0.5-5%.

According to the invention, in the anti-human IgG labeled by the chemiluminescence marker, the anti-human IgG is mouse anti-human IgG or sheep anti-human IgG, and the labeling molar ratio of the anti-human IgG antibody to the chemiluminescence marker is preferably 1: 3-20. The chemiluminescent label is preferably acridinium ester, acridinium ester derivatives and ruthenium terpyridyl.

According to the invention, the project diluent is a buffer solution which contains a surfactant, a preservative and protein and has a pH value of 6.0-8.0; wherein the buffer salt can be citrate, Bistris propane or phosphate, and is more preferably phosphate. The surfactant is 0.02-0.05% of Tween 20 or 0.05-0.1% of choline chloride; the preservative is 0.01-0.03% of sodium azide or 0.01-0.03% of PC300, and the protein is 0.1-3% of bovine serum albumin or 0.01-0.2% of bovine gamma globulin.

According to the invention, the chemiluminescent substrate solution comprises solution A and solution B, wherein the solution A is hydrogen peroxide and nitric acid solution, and the solution B is sodium hydroxide solution.

According to the invention, the calibrator is a Toxoplasma IgG calibrator.

Toxoplasma gondii IgG antibody solutions with the standard concentrations of Toxoplasma gondii IgG antibody of 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, and 200U/mL.

preparation of calibrator

Diluting TOXO IgG antibody into calibrator with 100mM PBS buffer containing 20% calf serum, and subpackaging into 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, 200U/mL;

second, preparing the solution of the magnetic bead coated antigen R1

And (3) fully and uniformly mixing the magnetic beads by using a vortex mixer, and adding 0.5-1.5 mL of suspended liquid (10mg/mL of magnetic beads) into a centrifuge tube. Placing the centrifugal tube on a magnetic separation frame for 1-3 min, carefully removing supernatant, adding 1mL of sealing agent, fully mixing by using a vortex mixer, then placing the centrifugal tube on the magnetic separation frame for 1-3 min (or longer), carefully removing supernatant, repeating the sealing step for 3 times, adding 1mL of sealing agent again, fully mixing by using the vortex mixer, adding 50-150 mu g of coating antibody (adding 100 mu L of antibody with equivalent concentration of 1 mg/mL), fully mixing by vortex, and incubating for 2-4 h by using a 360-degree rotary mixer for room-temperature rotary mixing. Placing the centrifugal tube on a magnetic separation frame for 1-3 min, carefully removing supernatant, adding 1mL of cleaning buffer solution, fully mixing by using a vortex mixer, placing the centrifugal tube on the magnetic separation frame for 1-3 min, and carefully removing supernatant. And (4) repeatedly washing for 3 times, using a storage buffer solution to resuspend the magnetic beads, collecting and storing at 2-8 ℃ for subsequent experiments.

Preparation of acridinium ester labeled anti-human IgG monoclonal antibody R2 solution

Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging for 20-40 s at room temperature by a centrifuge), adding a carbonic acid buffer solution, fully and uniformly mixing, adding 0.75-5 mu L of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging for 20-40 s at room temperature by the centrifuge. And sealing the centrifuge tube by using a sealing film, putting the centrifuge tube into a light-proof cassette, putting the cassette into a gas bath constant temperature oscillator (25 ℃), and uniformly mixing for 2-4 h. Adding 1mL of 20% lysine confining liquid, putting into a gas bath constant temperature oscillator (25 ℃), and uniformly mixing at medium speed for 0.5-2 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And (4) storing the collected antibody solution at 2-8 ℃. When in use, the purified anti-human IgG antibody concentrated solution is diluted by a buffer solution of 50mM MES, 0.05% Tween, 0.05% Proclin300 and pH 6.0-7.0 until the final concentration is 0.1-1.0 mu g/mL, and is stored at 2-8 ℃.

Fourthly, the project diluent R3

The R3 solution is pH6.0-8.0 buffer solution containing salt, surfactant, antiseptic and protein.

The three-phase reagent is encapsulated in different reagent bottles to form the reagent kit. The chemiluminescence immunoassay kit can complete the detection of Toxoplasma gondii IgG by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool. The chemiluminescence immunoassay kit is matched with an instrument, so that the time required by clinical detection is shortened. The chemiluminescence immunoassay kit has high detection precision.

When the kit is used for detecting the Toxoplasma gondii IgG antibody, a Dirui full-automatic chemiluminescence immunoassay analyzer (CM180) is used for detecting a Toxoplasma gondii IgG antibody calibrator, a standard curve is drawn, and the standard curve is built in computer software; then testing clinical samples according to requirements, and calculating the concentration of Toxoplasma gondii IgG antibody according to Relative Light Units (RLU) detected by the samples; and finally, evaluating the performance of the toxoplasma gondii IgG antibody chemiluminescence immunoassay kit.

In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.

Example 1

1) Preparation of calibrator

The TOXO IgG antibody was diluted with 100mM PBS buffer containing 20% calf serum to give a calibrator and dispensed at 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, 200U/mL.

2) Preparation of solution of magnetic bead coated antigen R1

The beads were mixed well using a vortex mixer and 0.5mL of the suspended liquid (10mg/mL of beads) was added to the centrifuge tube. The centrifuge tubes were placed on a magnetic separation rack for 1min and the supernatant carefully removed. Add 1mL of blocking agent, mix well using a vortex mixer, then place the centrifuge tube on a magnetic separation rack for 1min (or longer), carefully remove the supernatant. The blocking step was repeated a total of 3 times. Add 1mL of blocking agent again and mix well using vortex mixer. Add 50. mu.g of coating antibody and vortex well to mix. And (3) performing rotary mixing incubation for 2h at room temperature by using a 360-degree rotary mixer. The centrifuge tubes were placed on a magnetic separation rack for 1min and the supernatant carefully removed. Add 1mL of wash buffer and mix well using a vortex mixer. The centrifuge tubes were placed on a magnetic separation rack for 1min and the supernatant carefully removed. The washing step was repeated 3 times. And (4) resuspending the magnetic beads by using a storage buffer solution, collecting and storing at 2-8 ℃ for subsequent experiments.

3) Preparation of acridinium ester-labeled anti-human IgG monoclonal antibody R2 solution

Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging at room temperature for 20S) and then adding carbonic acid buffer solution, fully and uniformly mixing, adding 0.75 mu L of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging at room temperature for 20S by using a centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 2 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 deg.C), mixing at medium speed, and blocking for 0.5 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And (4) storing the collected antibody solution at 2-8 ℃. When in use, the purified anti-human IgG antibody concentrated solution is diluted by a buffer solution of 50mM MES, 0.05% Tween, 0.05% Proclin300 and pH6.0 to a final concentration of 0.1 mu g/mL, and is stored at 2-8 ℃.

4) Preparation of project dilution R3 solution

The solution of R3 is a buffer solution with pH6.0 containing citric acid, 0.02% Tween 20, 0.01% sodium azide, 0.01% PC300, 0.1% bovine serum albumin and 0.01% bovine gamma globulin.

Example 2

1) Preparation of calibrator

2) Preparation of magnetic bead coated antigen R1 solution

The beads were mixed well using a vortex mixer and 1mL of the suspended liquid (10mg/mL of beads) was added to the centrifuge tube. The centrifuge tubes were placed on a magnetic separation rack for 2min and the supernatant carefully removed. Add 1mL of blocking agent, mix well using a vortex mixer, then place the centrifuge tube on a magnetic separation rack for 2min (or longer), carefully remove the supernatant. The blocking step was repeated a total of 3 times. Add 1mL of blocking agent again and mix well using vortex mixer. Add 100. mu.g of coating antibody and vortex well to mix. And (3) performing rotary mixing incubation for 3h at room temperature by using a 360-degree rotary mixer. The centrifuge tubes were placed on a magnetic separation rack for 2min and the supernatant carefully removed. Add 1mL of wash buffer and mix well using a vortex mixer. The centrifuge tubes were placed on a magnetic separation rack for 2min and the supernatant carefully removed. The washing step was repeated 3 times. And (4) resuspending the magnetic beads by using a storage buffer solution, collecting and storing at 2-8 ℃ for subsequent experiments.

Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging the antibody at room temperature for 30S) and then adding carbonic acid buffer solution, fully and uniformly mixing, adding 2.5 mu L of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging the mixture at room temperature for 30S by using a centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 3 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 ℃), and mixing at medium speed for 1 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And (4) storing the collected antibody solution at 2-8 ℃. When in use, the purified anti-human IgG antibody concentrated solution is diluted to a final concentration of 0.5 mu g/mL by using a buffer solution of 50mM MES, 0.05% Tween, 0.05% Proclin300 and pH6.5, and is stored at 2-8 ℃.

4) Preparation of project dilution R3 solution

The solution R3 is a buffer solution with pH7.0 containing Bistris propane, 0.05% choline chloride, 0.02% sodium azide, 0.02% PC300, 1% bovine serum albumin and 0.1% bovine gamma globulin.

Example 3

1) Preparation of calibrator

2) Preparation of magnetic bead coated antigen R1 solution

The beads were mixed well using a vortex mixer and 1.5mL of the suspended liquid (10mg/mL of beads) was added to the centrifuge tube. The centrifuge tubes were placed on a magnetic separation rack for 3min and the supernatant carefully removed. Add 1mL of blocking agent, mix well using a vortex mixer, then place the centrifuge tube on a magnetic separation rack for 3min (or longer), carefully remove the supernatant. The blocking step was repeated a total of 3 times. Add 1mL of blocking agent again and mix well using vortex mixer. Add 150. mu.g of coating antibody and vortex well to mix. And (4) performing rotary mixing incubation for 4h at room temperature by using a 360-degree rotary mixer. The centrifuge tubes were placed on a magnetic separation rack for 3min and the supernatant carefully removed. Add 1mL of wash buffer and mix well using a vortex mixer. The centrifuge tubes were placed on a magnetic separation rack for 3min and the supernatant carefully removed. The washing and the steps were repeated 3 times. And (4) resuspending the magnetic beads by using a storage buffer solution, collecting and storing at 2-8 ℃ for subsequent experiments.

Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging the antibody at room temperature for 40S), adding carbonic acid buffer solution, fully and uniformly mixing, adding 5 mu L of 2mg/mL of acridinium ester DMF solution after uniformly mixing, and centrifuging the mixture at room temperature for 40S by using a centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 4 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 ℃), and mixing at medium speed for 2 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And (4) storing the collected antibody solution at 2-8 ℃. When in use, the purified anti-human IgG antibody concentrated solution is diluted to a final concentration of 1.0 mu g/mL by using a buffer solution of 50mM MES, 0.05% Tween, 0.05% Proclin300 and pH7.0, and is stored at 2-8 ℃.

4) Preparation of project dilution R3 solution

The solution of R3 is a buffer solution with pH8.0 containing phosphate, 0.05% Tween 20, 0.03% sodium azide, 0.03% PC300, 3% bovine serum albumin and 0.2% bovine gamma globulin.

Example 4 Toxoplasma gondii IgG antibody chemiluminescence immunoassay kit principle and detection method

The detection principle of the kit is an indirect method, the sample volume is 20 mu L, the antigen reagent volume coated by magnetic beads is 40 mu L, the project diluent reagent volume is 50 mu L, and the acridinium ester labeled anti-human IgG antibody reagent volume is 50 mu L.

Firstly, adding 10 mu L of sample, adding 90 mu L of diluent, diluting by 10 times, taking 20 mu L of diluted sample, then respectively adding an antigen R1 solution coated by magnetic beads and a buffer R3, incubating for 18min, washing, adding an acridinium ester labeled antibody R2 reagent, incubating for 5min to form a magnetic microsphere-TOXO antigen-TOXO antibody-anti-human IgG antibody-acridinium ester compound, separating the immune compound by a magnetic separator, and washing off free components. Relative Light Units (RLU) are recorded by exciting acridinium ester to emit light through an acid-base excitation liquid, and the content of the TOXO IgG antibody in the sample is in direct proportion to the relative light units in a linear range.

Example 4: and (3) preparing the performance evaluation of the TOXO IgG antibody chemiluminescence detection kit.

1. Kit linearity assay

Using 1 lot of the kit prepared in example 1, calibration curves were prepared using 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, and 200U/mL of the calibration sample, and as shown in FIG. 1, the linear range was 0.1U/mL-200U/mL, and the linear correlation coefficient r was not less than 0.9986.

TABLE 1 kit Linear Range determination

2. Determination of sensitivity of kit

Using 1 batch of the kit prepared in example 1, 20 times of zero concentration samples were measured, the mean (M) and Standard Deviation (SD) were calculated to obtain M +2SD, two-point regression fitting was performed according to the concentration-RLU between the zero concentration calibrator and the adjacent calibrator to obtain a linear equation, and the RLU of M +2SD was substituted into the equation to obtain the corresponding concentration value, i.e., the kit sensitivity was 0.0141U/mL.

TABLE 2 determination of sensitivity of the kit

3. Precision test of kit

(1) Precision in batch

The high value sample (150U/mL) and the low value sample (12U/mL) were each assayed 10 times in 1 of the kits prepared in example 1, giving a coefficient of variation in batch of < 8%.

TABLE 3 results of in-batch precision measurement

Sample concentration (U/mL)	Number of measurements	Coefficient of variation CV%
			12	10	4.41
150	10	2.23

(2) Inter-batch precision

The kit prepared in example 1 was used, wherein 3 batches of the kit were repeatedly measured 10 times for each of the high value sample (150U/mL) and the low value sample (12U/mL), and the average and standard deviation of the 30 measurements were calculated to obtain a coefficient of variation between batches of less than 15%.

TABLE 4 results of measurement of precision between lots

Sample concentration (U/mL)	Determination of the Total number of times	Coefficient of variation CV%
			12	30	7.12
150	30	3.47

4. Determination of accuracy of kit

Using 1 batch of the kit prepared in example 1, labeled with a standard substance at a concentration of 20IU/mL, the relative deviation was < 10%, and the test results were as follows:

TABLE 5 results of accuracy determination

Concentration of Standard substance (U/mL)	Measurement results	Relative deviation%
			20	20.57	2.85％

5. Anti-interference and specificity experiment of kit

An interference sample containing bilirubin (40mg/dL), hemoglobin (2g/dL) and triglyceride (2000mg/dL) is subjected to a recovery experiment, and the detection result is not interfered (the interference rate is less than or equal to 10%); the specific samples (BV antibody, HCV antibody, HIV antibody, CMV antibody, EBV antibody, HSV antibody, rubella virus antibody and treponema pallidum antibody positive samples in clinical detection) are tested, the detection result is not influenced, and the test result is as follows:

TABLE 6 results of anti-interference experiments

Potentially interfering substances	Theoretical concentration (mg/dL)	Test value (mg/dL)	Average recovery (%)
				Bilirubin	40	41.27	103.18
Hemoglobin	2000	1948.2	97.41
				Triglycerides	2000	2042.6	102.13

TABLE 7 results of specificity measurement

Specific sample	Clinical determination of the interferent results	Toxoplasma IgG antibody detection results
			BV antibody	Positive for	Negative of
HCV antibodies	Positive for	Negative of
			HIV antibodies	Positive for	Negative of
CMV antibodies	Positive for	Negative of
			Rubella virus antibody	Positive for	Negative of
EBV antibodies	Positive for	Negative of
			HSV antibodies	Positive for	Negative of
Treponema pallidum	Positive for	Negative of

Claims

1. A chemiluminescence immunoassay assay kit for detecting Toxoplasma gondii IgG antibody is characterized by comprising:

2. The chemiluminescence immunoassay kit for detecting toxoplasma gondii IgG antibody according to claim 1, wherein the particle size of the magnetic bead in the TOXO antigen coated by the magnetic bead is 0.05-3.0 μm, and the concentration of the TOXO antigen coated by the magnetic bead is 0.5% -5%.

3. The chemiluminescence immunoassay assay kit for detecting Toxoplasma gondii IgG antibody according to claim 1, wherein the anti-human IgG labeled with the chemiluminescent label is mouse anti-human IgG or goat anti-human IgG.

4. The chemiluminescence immunoassay kit for detecting toxoplasma gondii IgG antibody according to claim 1, wherein the labeling molar ratio of the anti-human IgG antibody to the chemiluminescent label is 1: 3-20.

5. The chemiluminescent immunoassay assay kit for the detection of Toxoplasma gondii IgG antibodies of claim 1, wherein said chemiluminescent label is acridinium ester, acridinium ester derivatives and ruthenium terpyridyl.

6. The chemiluminescence immunoassay kit for detecting toxoplasma gondii IgG antibody according to claim 1, wherein the item diluent is a buffer solution with pH6.0-8.0 and containing salt, surfactant, preservative and protein.

7. The chemiluminescence immunoassay kit for detecting toxoplasma gondii IgG antibody according to claim 1, wherein the chemiluminescence excitation liquid a is hydrogen peroxide and nitric acid solution, and B is sodium hydroxide solution; the cleaning solution is PB buffer solution.

8. The chemiluminescent immunoassay assay kit for the detection of Toxoplasma IgG antibodies of claim 1, wherein the calibrator is formulated with TOXO IgG antibodies.

9. The method for preparing the chemiluminescence immunoassay assay kit for detecting the Toxoplasma gondii IgG antibody according to claim 1, characterized in that the method comprises the following steps:

preparation of calibrator

second, magnetic bead coated TOXO antigen solution preparation

four, project diluent