CN104597250A - Latex enhanced immune turbidimetric kit for detecting content of human replication protein variants - Google Patents

Abstract

The invention discloses a latex-enhanced immune turbidimetric kit for detecting the content of human replication protein variants. The kit includes reagent R1, reagent R2 and replication protein variant standards, wherein reagent R1 contains electrolytes, coagulants, preservatives and buffers; reagent R2 contains polyclonal antibody-coated latex against human replication protein variants Particles, electrolytes, stabilizers, preservatives, and buffers; standards are appropriate buffers containing varying concentrations of replicating protein variants. The kit has strong specificity, high sensitivity, good accuracy, quick and easy operation, and can be used in various biochemical analyzers.

Description

A latex-enhanced immunoturbidimetric kit for detecting the content of human replication protein variants

technical field

The invention belongs to the field of in vitro diagnostic reagents for medical immunity, and relates to a latex-enhanced immune turbidimetric kit for detecting the content of human replication protein variants.

Background technique

Human replication protein CIZ1 ( C ip1 interacting z inc finger protein 1 ) is a nucleoprotein involved in DNA replication, which is associated with many human diseases including various cancers. Studies have shown that the CIZ1 mutant (CIZ1-V) with mutations in exons 14 and 15 is an early lung cancer and a variety of tumor biomarkers, and it can be used as a single marker for tumor-specific detection without relying on traditional medicine. An invasive test to diagnose and identify patients with early-stage lung cancer from at-risk populations. Since the polypeptide sequence CDEDEEEIEVRSRDISR of the mutant fragment of the current CIZ1 mutant (CIZ1-V) is very short, general detection requires gene sequencing or invasive detection methods to be detected by techniques such as PCR.

Contents of the invention

The purpose of the present invention is to provide a latex-enhanced immune turbidimetric kit for detecting the content of human replication protein variants aiming at the above-mentioned defects of the prior art.

The purpose of the present invention can be achieved through the following technical solutions:

A latex-enhanced immunoturbidimetric kit for detecting the content of human replication protein variants, comprising reagent R1, reagent R2 and replication protein variant standard solution, wherein:

The reagent R1 includes electrolytes, coagulation accelerators, preservatives and buffers;

The reagent R2 comprises polyclonal antibody-coated latex particles against human replication protein variants, electrolytes, stabilizers, preservatives and buffers;

The replicator variant standard solution is a buffer solution containing replicator variant standard products at different concentrations.

Preferably, the reaction buffer is one or more of PBS buffer, Hepes buffer, MES buffer, Tris-HCl buffer, glycine buffer, acetate buffer or DIPSO buffer, and the buffer concentration Between 20-200mM.

Preferably, the preservative in R1 is preferably sodium azide (NaN ₃ ), the coagulant is selected from PEG6000, PEG8000 and PEG12000, preferably PEG6000 or a mixture of different PEGs, and the concentration in reagent R2 is 2.0%-3.5% .

Preferably, the latex particles are polystyrene latex particles with a diameter in the range of 0.05-0.20 μm, and the concentration of the latex particles in the reagent R2 is 0.1%-0.8%.

Further preferably, the latex particles are polystyrene latex particles with a diameter of 0.13 μm, and the concentration of the latex particles in the reagent R2 is 0.2%.

The polyclonal antibody against human replication protein variants is selected from rabbit, sheep, and chicken immunoglobulins, preferably rabbit antibodies, immunoglobulins obtained through human replication protein variant immunization and affinity chromatography purification; more preferably Synthesize the mutant fragment polypeptide sequence CDEDEEEIEVRSRDISR (SEQ ID NO.1) expressed by exons 14 and 15 of CIZ1-V, couple the synthesized polypeptide sequence to hemocyanin carrier protein to prepare antigen and immunize rabbits, sheep, and chickens , and then purify the obtained immunoglobulin by affinity chromatography.

Coating of latex particles with polyclonal antibodies against human replicatin variants by chemical crosslinking agents such as carbodiimide (EDAC) and N-hydroxysuccinimide in crosslinking buffer (NHS), the cross-linking buffer is selected from PBS buffer, Hepes buffer, MES buffer, Tris-HCl buffer, glycine buffer, acetate buffer or DIPSO buffer, pH between 6.0-8.0.

Preferably, the polyclonal antibody of the anti-human replication protein variant utilizes chemical cross-linking agent carbodiimide (EDAC) and N-hydroxyl succinimide (NHS) The way of directional immobilization on the styrene latex particle is: Take 20 μg of latex microspheres in a 1.5mL EP tube, add 1mL of MES buffer (pH6.0), mix with the tip of the pipette; centrifuge at 14000rpm for 30min, remove the supernatant, add 0.5mL of LEDAC solution (0.2-2.0mg/mL), 22 Rotate at 16 rpm for 10 min; then add 50 μL NHS solution (0.2-2.0 mg/mL), rotate at 16 rpm at 22 °C for 30 min, the latex particles are activated, then centrifuge at 14000 rpm for 30 min, remove the supernatant, add 1 mL of 50 mMPBS buffer (pH7.4), Mix the tip of the pipette; centrifuge at 14000rpm for 30min, remove the supernatant, add 1mL of 50mM PBS buffer (pH7.4), sonicate for 15s, add 20μg of CIZ1-V antibody, rotate at 16rpm at 37°C for 2h; add 10μL of stop solution (1M glycine + 10 %BSA), rotate at 16rpm at 37°C for 1h. Centrifuge at 14000rpm for 25min, remove the supernatant, add 1mL storage solution (30mM pH7.4 Hepes or DIPSO buffer, preservative 0.1% NaN ₃ , electrolyte 0.01% NaCl, stabilizer 0.01% EDTANa ₂ and 0.09% BSA), and sonicate for 20s; Centrifuge at 14000rpm for 20min, remove the supernatant, add 1mL of storage solution, sonicate for 20s, and store at 4°C.

The said replication protein variant standard product is prepared by the following method: synthesize the mutant fragment polypeptide sequence CDEDEEEIEVRSRDISR expressed by exon 14 and 15 of CIZ1-V, and couple the synthesized polypeptide to ovalbumin (OVA) carrier protein to obtain replication Protein variant standards.

Preferably, the replication protein variant standard solution is an appropriate buffer containing different concentrations of replication protein variant standards, wherein the replication protein variant content is 0 μg/mL, 0.50 μg/mL, 1.00 μg/mL, 2.00 μg/mL, respectively. μg/mL, 4.00μg/mL, 8.00μg/mL or similar ratio concentration, the buffer is one or more of PBS buffer, Hepes buffer or Tris-HCl buffer.

Preferably, 112.5 μL and 37.5 μL are added to R1 and R2 respectively during detection, and the sample addition volume is 2-8 μL, or an overall increase or decrease in a similar proportion.

Most preferably, the latex-enhanced immune turbidimetric kit for detecting the content of replication protein variants in human serum consists of the following:

Reagent R1: 50mM PBS buffer (pH7.4), 2.5% PEG8000, 0.015% NaCl, 0.5% NaN ₃ ;

Reagent R2: 0.2% rabbit anti-human replicatin variant antibody coated latex particles (particle size 0.13 μm), 30 mM Hepes or DIPSO buffer at pH 7.4, 0.1% NaN ₃ , 0.01% NaCl, 0.01% EDTANa ₂ and 0.09 %BSA;

Replicate protein variant standard solution: the synthetic CIZ1-V exon 14, 15 expressed polypeptide sequence CDEDEEEIEVRSRDISR coupled ovalbumin (OVA) carrier protein as antigen standard, 50mM PBS buffer (pH7.4).

In the present invention, antigen-antibody reaction occurs between the replica protein variant and the rabbit anti-human replica protein variant polyclonal antibody coated with latex particles to form an antigen-antibody-latex particle complex, which causes the turbidity to rise. Preferably, the The absorbance of the turbidity at a wavelength of 500-600nm (most preferably, 600nm) can be compared with a standard curve to calculate the replicative protein variant content in the sample.

Beneficial effect:

At present, there are very few reports on CIZ1-V, and there are no related kit technologies and products available. Since the polypeptide sequence CDEDEEEIEVRSRDISR of the mutant fragment of the current CIZ1 mutant (CIZ1-V) is very short, general detection requires gene sequencing or invasive detection methods to be detected by techniques such as PCR. In the present invention, the mutant fragment polypeptide sequence CDEDEEEIEVRSRDISR expressed by exons 14 and 15 of CIZ1-V is coupled to the hemocyanin carrier protein to prepare an antigen, which is obtained by immunizing rabbits, sheep and chickens, and then purified by affinity chromatography Immunoglobulin. Synthesize the polypeptide sequence CDEDEEEIEVRSRDISR of the mutant fragment expressed by exons 14 and 15 of CIZ1-V, and couple the synthesized polypeptide to the ovalbumin (OVA) carrier protein to obtain a replica protein variant standard, so that the small mutant fragment can also be used in conventional The latex-enhanced immunoturbidimetric kit can be tested on an automatic biochemical detector by optimizing the conditions, achieving low-cost, convenient, fast and correct clinical testing.

The present invention coats the specific rabbit anti-human replication protein variant antibody on the surface of latex particles obtained after activation by chemical coupling method, and makes the latex particles form a uniform and stable suspension by adding specific stabilizers, preservatives and coagulants. Cloudy liquid. It is easy and quick to operate, and meets the requirements of clinical rapid and high-throughput screening. The use of specific antibody immunoglobulin ensures the high specificity of the kit and effectively avoids the interference of other substances.

The performance identification of the present invention selects several parameters of sensitivity, accuracy, precision and specificity, and compares the detection results with the Elisa method. The correlation coefficient ^R2 of the standard curve of this kit reaches 0.9988, the linearity is good, the detection limit reaches 0.2 μg/mL, the recovery rate of the target protein obtained from expression and purification is relatively high, and the coefficient of variation is low, and the coefficient of variation between batches and within batches All are less than 10%, indicating that the kit has good accuracy and precision. Through the comparative analysis of the detection results of this kit and the Elisa method, it was shown that there was no significant difference in the recovery rate of the target protein obtained by the expression and purification of the two methods for the same concentration (P≥0.05). There is no significant difference in the results when the two methods are used to detect protein expression (P≥0.05), but the operation time is shorter than the Elisa method, simple and convenient, and low cost. This kit can specifically and accurately detect the content of the target protein from various proteins, indicating that it has high specificity and is convenient for rapid detection.

Description of drawings

Figure 1 Standard curve of detection concentration of Replication Protein Variant Latex Enhanced Immunoturbidity Kit

Figure 2 SDS-PAGE identification of CIZ1-V fusion protein expression and purification results

Among them, M: protein marker; 1: supernatant before column; 2: supernatant after column; 3: target protein

Figure 3 Comparison of detection results after reaction between R2 and R1 prepared with different particle sizes of latex microspheres and standard products

Figure 4 Comparison of detection results of R2 prepared from 0.13 μm latex at different latex concentrations

Figure 5 0.13μm latex with 0.2% concentration is prepared into R2 and compares the detection results at different wavelengths

Detailed ways

In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Preparation of Replication Protein Variant Latex Enhanced Immunoturbidity Kit

Reagent R1: Reagent R1: 50 mM PBS buffer (pH7.4), 2.5% PEG8000, 0.015% NaCl, 0.5% NaN ₃ , the reagent is a colorless transparent solution.

Reagent R2: 0.2% rabbit anti-human replicatin variant antibody-coated latex particles (particle size: 0.13 μm), 30 mM Hepes or DIPSO buffer at pH7.4, 0.1% NaN ₃ , 0.01% EDTANa ₂ and 0.09% BSA;

Preparation:

(1) Take 0.2mg of latex microspheres with a particle size of 0.099μm, 0.13μm, 0.189μm, and 0.20μm in a 50mL centrifuge tube, add 10mL of MES buffer (pH6.0), mix with the tip of the pipette, and centrifuge at 14000rpm for 30min. to the supernatant;

(2) Weigh 2 mg of EDAC powder and dissolve it in 10 mL of MES buffer (pH6.0) to prepare 0.2 mg/mL EDAC solution, add 5 mL to the above centrifuge tube, and rotate at 22 ° C for 10 min at 16 rpm;

(3) Weigh 8 mg of NHS powder and dissolve it in 1 mL of MES buffer (pH 6.0) to prepare 8 mg/mL NHS solution. Take 0.5 mL and add it to the above centrifuge tube. After dissolving and mixing, rotate at 16 rpm for 30 min at 22 ° C to activate the latex particles. Centrifuge at 14000rpm for 30min, remove the supernatant;

(4) Add 10mL 50mM PBS buffer solution (pH7.4) to the above centrifuge tube, mix with the pipette tip; centrifuge at 14000rpm for 30min, remove the supernatant;

(5) Add 10mL of 50mM PBS buffer solution (pH7.4) to the above centrifuge tube, mix with ultrasound, add 0.2mg of CIZ1-V polyclonal antibody, and rotate at 16rpm at 37°C for 2h to make the antibody and latex undergo coupling reaction;

(6) Add 0.1 mL of stop solution (1M glycine + 10% BSA) to the above-mentioned centrifuge tube, rotate at 16 rpm at 37°C for 1 hour, and carry out the stop reaction;

(7) centrifuge at 14000rpm for 25min, remove the supernatant;

(8) Add reagent 30mM Hepes or DIPSO buffer at pH7.4, preservative 0.1% NaN ₃ , electrolyte 0.01% NaCl, stabilizer 0.01% EDTANa ₂ and 0.09% BSA into the above centrifuge tube; ultrasonically mix; centrifuge at 14000rpm 20min, remove the supernatant;

(9) Add 10mL DIPSO buffer solution, 0.1% NaN ₃ preservative, 0.01% NaCl electrolyte, 0.01% EDTANa ₂ stabilizer and 0.09% BSA into the above centrifuge tube, and obtain reagent R2 after ultrasonic mixing. save.

Replication protein variant standard solution: in 50mM PBS buffer (pH7.4), add the mutant fragment polypeptide sequence CDEDEEEIEVRSRDISR of the synthetic CIZ1-V exon 14 and 15 gene expression coupled to ovalbumin (OVA) carrier protein , as replica protein variant standards, the final concentrations were 0 μg/mL, 0.50 μg/mL, 1.00 μg/mL, 2.00 μg/mL, 4.00 μg/mL, 8.00 μg/mL. And use this as a calibration to carry out relative quantitative detection of samples.

Example 2 The detection steps of the replication protein variant latex enhanced immune turbidimetric kit and the drawing of a standard curve

Detection wavelength: 600nm;

Sample volume: 8 μL; R1: 250 μL; R2: 75 μL; the composition of R1 and R2 is the same as in Example 1.

Taking the Hitachi 7080 biochemical analyzer as an example, first add R1, incubate at 37°C for 30s, then add the standard (sample to be tested), incubate for 5min, then add R2, measure the absorbance values at the 10s and 5min of the reaction (respectively A ₁ , A ₂ ), calculate the absorbance difference OD ₆₀₀ =A ₂ -A ₁ ;

Draw the standard curve: take the concentration of the standard substance as the abscissa, and the absorbance difference OD ₆₀₀ as the ordinate to draw the detection concentration standard curve and establish a regression equation to obtain the calibration curve, as shown in Figure 1;

Calculation method: Substituting the measured absorbance difference OD ₆₀₀ of the samples to be tested into the regression equation, the concentration of the replicator variant in the samples to be tested can be calculated.

Example 3 Detection sample preparation:

1. CIZ1-V expression plasmid construction and fusion protein expression and purification:

Design PCR primers according to the gene sequence information of exons 14 and 15 of CIZ1-V:

CIZ1-V-pet-F: 5'-CCG GAATTC CATCATCATCACCATCATCTG-3' (SEQ ID NO.3),

CIZ1-V-pet-R: 5'-CCC AAGCTT CTCGAG TTAATATGCGGTATTCGGGC-3' (SEQ ID NO.4),

Among them, restriction sites are underlined. Using the nucleotide TGTGATGAGGATGAAGAAGAGATCGAGGTGAGGTCCAGAGATATATCCAGA (SEQ ID NO.2) of the mutated fragment polypeptide sequence CDEDEEEIEVRSRDISR of the synthesized CIZ1-V exon 14 and 15 gene expression as a template, PCR amplification was carried out, and the reaction conditions were: 95°C pre-denaturation for 5 minutes; Cycle 30 times according to the following parameters: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, and finally extension at 72°C for 10 min. PCR reaction products were analyzed by 1% agarose gel electrophoresis. After the target gene was amplified by PCR, the PCR product was separated and purified by gel electrophoresis, the target gene was recovered from the gel, and the expression vector pGEX-2T was digested with EcoRI and HindIII, respectively, and ligated with the target gene. The ligation product was transferred into E.coli DH5α, the sequence of the target gene was determined, and the sequencing results were compared with the sequences of exons 14 and 15 of CIZ1-V by N-BLAST sequence analysis on NCBI.

The recombinant plasmid with the correct sequence was extracted and transformed into E.coli BL21(DE3). Inoculate a single colony containing the expression plasmid in LB medium (1L) containing ampicillin (100μg/mL), induce expression with IPTG (1mmol/L) at 37°C for 4h, centrifuge at 8000rpm for 15min, remove the supernatant, and add 50mL PB lysate, lysed at 4°C for 0.5h, sonicated (30s, 30s, 10min) in an ice bath and stored at -20°C.

After ultrasonication, the sample was centrifuged at 11000 rpm for 15 min, and the supernatant was purified by GST affinity purification method (column passing method), the impurity protein was washed with reducing glutamic acid solution, and the target protein was washed with PBS (pH7. The pre-column supernatant, post-column supernatant and the obtained target protein were analyzed by SDS-PAGE, as shown in Figure 2.

A total of 4 mL of the target protein solution was obtained, and the OD value at 595 nm was measured using Protein Assay as a dye to draw a BSA concentration standard curve, and the protein concentration was calculated to be 0.636 mg/mL according to the OD ₅₉₅ value of the purified target protein and the regression equation obtained from the standard curve.

2. Dilute the CIZ1-V target protein obtained by expression and purification with PBS buffer, so that the final concentrations are 1 μg/mL, 2 μg/mL, 4 μg/mL, and 8 μg/mL, which are used as the identification accuracy of the kit of the present invention, Precision and samples compared with the detection results of the Elisa method.

3. Take an appropriate amount of PB lysate (total 50 mL) of CIZ1-V expressing bacteria after sonication, and dilute it 200 times, which is equivalent to the original volume of the expressing bacterial liquid. Used as a detection sample for the preliminary application of the kit of the present invention.

Example 4, Screening of Replication Protein Variant Latex Enhanced Immunoturbidimetric Kit Components

The prepared R1 and R2 were screened using the Hitachi 7080 biochemical analyzer as an example. First, add R1, incubate at 37°C for 30s, then add the standard (sample to be tested), incubate for 5min, then add R2, and measure React the absorbance values at 10 s and 5 min (respectively A ₁ and A ₂ ), and calculate the absorbance difference OD ₆₀₀ =A ₂ -A ₁ .

The absorbance difference detected by latex with different particle sizes is shown in Figure 3, and R2 prepared from latex with a linear relationship of 0.13 μm is preferred.

When other conditions are the same, preferably 0.13 μm latex prepared R2, under different latex concentrations, carry out the same detection, the results are shown in Figure 4, the preferred 0.2% concentration of latex is prepared to make R2 the best linear relationship within the detection range.

After determining the concentration of latex in R2, perform the optimum detection wavelength. The results are shown in Figure 5. When other conditions are the same, select 4 common wavelengths between 500-600nm for detection. The larger the wavelength, the greater the discrimination, and the linearity There is not much difference in the relationship. Relatively speaking, the linear relationship at 600nm is the best within the detection range, so this wavelength is selected for the detection of the kit.

Example 5 Replication protein variant latex enhanced immune turbidimetric kit performance detection

This embodiment investigates the performance of the kit based on the detection conditions optimized in Example 4

Statistical analysis: data expressed as mean ± standard error SPSS 19.0 software was used for single-factor independent T-test analysis. P<0.05 was judged as significant difference, and P≥0.05 was judged as insignificant difference.

1. Sensitivity detection:

Measure the blank standard substance of 20 different batches with the kit of the present invention, calculate the average value of _OD600 value and standard deviation(s), sensitivity The concentration calculated according to the standard curve regression equation is the theoretical detection limit of the kit. As shown in Table 1 below, by substituting the calculated sensitivity into the calibration curve, the theoretical detection limit of the kit of the present invention can be calculated to be 0.11 μg/mL. Considering the operational error, the detection limit of the kit is determined to be 0.20 μg/mL.

Table 1 Sensitivity detection of the kit of the present invention

0μg/mL 1 0.0456 2 0.0289 3 0.0398 4 0.0424 5 0.0331 6 0.0415

7 0.0449 8 0.0381 9 0.0357 10 0.0432 11 0.0463 12 0.0405 13 0.0462 14 0.0482 15 0.0395 16 0.0387 17 0.0474 18 0.0393 19 0.0504 20 0.0454 average value 0.0418 standard deviation 0.0054 sensitivity 0.0310

2. Accuracy detection:

Use the prepared CIZ1-V expression and purification target protein solution as a test sample, the gradient concentrations are 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, and use the kit of the present invention for detection, each concentration is set to 6 The accuracy was determined by the recovery rate and coefficient of variation (CV) after repeated determination. The results in Table 2 show that the recovery rate of the CIZ1-V kit for the expressed and purified target protein ranges from 98.92% to 104.00%, with an average of 100.91%. The coefficients of variation are all less than 10%, indicating that the kit has high accuracy.

Table 2 Detection of the accuracy of the kit of the present invention

3. Precision testing:

Three batches of the kit of the present invention marked with different batches were taken, and the purified CIZ1-V target protein with a concentration of 1 μg/mL, 2 μg/mL, 4 μg/mL, and 8 μg/mL was measured respectively, and 6 repeated determinations were set for each concentration. The precision was determined by within-assay CV and between-assay CV. The results in Table 3 show that the CIZ1-V kit has a range of 95.32%-105.00% recovery of the target protein expressed and purified, with an average of 99.51%; the intra-assay coefficient of variation ranges from 1.13%-2.64%, with an average of 1.91%; inter-assay variation The coefficient ranges from 0.57% to 2.06%, with an average of 1.19%. The inter-assay and intra-assay coefficients of variation of the measured values are both less than 10%, indicating that the kit has high precision.

Table 3 Detection of kit precision of the present invention

4. Compared with the detection results of Elisa method:

The purified CIZ1-V protein with a concentration of 1 μg/mL, 2 μg/mL, 4 μg/mL and 8 μg/mL was measured respectively by using the kit of the present invention and an enzyme-linked immunosorbent assay (Elisa) method, and 6 repeated determinations were set for each concentration , and compare the two test results. The results in Table 4 show that compared with the Elisa method, there is no significant difference between the measured value of the kit of the present invention and the measured value of the Elisa method (P≥0.05), and there is no significant difference between the two methods for the recovery of the same concentration of the purified target protein (P≥0.05).

Table 4 kit of the present invention and Elisa method detection result comparison

Between the measured values of the same concentration, different shoulder marks indicate significant differences, P<0.05

Embodiment 6 Preliminary application detection effect of the kit of the present invention

Take the prepared protein expression solution and negative control, and use the developed kit and Elisa method to measure respectively, and set 3 repeated determinations. The results are shown in Table 5. The result shows that there is no significant difference (P≥0.05) between the CIZ1-V protein expression concentration measured by the test kit of the present invention and the Elisa method measured value, and the protein of interest can be detected from a large amount of miscellaneous proteins, showing that the test kit has relatively High specificity.

Table 5 Preliminary application detection effect of kit of the present invention and Elisa method detection result comparison

Between the measured values of the same concentration, different shoulder marks indicate significant differences, P<0.05

The above embodiments show and describe the basic principles, main features and advantages of the present invention. What described in above-mentioned embodiment and specification sheet just tells about the principle of the present invention, the present invention also can have various changes and improvements under the premise of not departing from the spirit and scope of the present invention, and these changes and improvements all fall into the claimed scope of the present invention Inside. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. A latex-enhanced immune turbidimetric kit for detecting human replication protein variant content, characterized in that the test kit includes reagent R1, reagent R2 and replication protein variant standard solution, wherein: The reagent R1 includes electrolytes, coagulation accelerators, preservatives and buffers; The reagent R2 comprises polyclonal antibody-coated latex particles against human replication protein variants, electrolytes, stabilizers, preservatives and buffers; The replicator variant standard solution is a buffer solution containing replicator variant standard products at different concentrations. 2. kit according to claim 1, it is characterized in that the polyclonal antibody of described anti-human replication protein variant is selected from the immunoglobulin of rabbit, sheep, chicken, preferably rabbit anti, through human replication protein variant The immunoglobulin obtained by immunization and affinity chromatography purification; further preferred to synthesize the mutant fragment polypeptide expressed by the CIZ1-V exon No. 14 and No. 15 genes shown in SEQ ID NO.1, and couple the synthetic polypeptide to blood The immunoglobulin obtained by preparing antigen on the blue protein carrier protein and immunizing rabbits, sheep, and chickens, and then purified by affinity chromatography. 3. The kit according to claim 1, characterized in that the latex particles are polystyrene latex particles with a diameter range of 0.05-0.20 μm, and the concentration of the latex particles in the reagent R2 is 0.1%-0.8%. 4. The kit according to claim 3, characterized in that the latex particles are polystyrene latex particles with a diameter of 0.13 μm, and the concentration of the latex particles in the reagent R2 is 0.2%. 5. kit according to claim 1, it is characterized in that the polyclonal antibody coating latex particle of anti-human replication protein variant is to carry out in cross-linking buffer by chemical cross-linking agent, used chemical cross-linking agent is Carbodiimide (EDAC) and N-hydroxysuccinimide (NHS), cross-linking buffer selected from PBS buffer, Hepes buffer, MES buffer, Tris-HCl buffer, glycine buffer, acetate buffer Or DIPSO buffer, pH between 6.0-8.0. 6. The kit according to claim 1, characterized in that the coagulant in the reagent R1 is selected from PEG6000, PEG8000, PEG12000 with a concentration range of 2.0%-3.5%, preferably 2.5% PEG6000 or a mixture of different PEGs body. 7. The test kit according to claim 1, wherein the electrolyte described in the reagent R1 is selected from sodium chloride, potassium chloride, magnesium chloride, and the concentration is 0.01%-1.5%; the electrolyte described in the reagent R2 is selected from From sodium chloride, potassium chloride, magnesium chloride, the concentration is 0.01%-1%. 8. The kit according to claim 1, wherein the preservatives described in reagents R1 and R2 are selected from sodium azide, and the concentration is 0.05%-0.1%; the buffer is selected from PBS buffer, One of Hepes buffer, MES buffer, Tris-HCl buffer, glycine buffer, acetate buffer or DIPSO buffer, the buffer concentration is between 20-200mM. 9. The kit according to claim 1, characterized in that the stabilizer in the reagent R2 is selected from bovine serum albumin and _EDTANa2 at a concentration of 0.1%-1.0%. 10. The kit according to claim 1, characterized in that said replication protein variant standard is prepared by the following method: Synthesizing the CIZ1-V Exon 14, No. 15 gene as shown in SEQ ID NO.1 The expressed mutant fragment polypeptide was coupled to the ovalbumin (OVA) carrier protein to obtain the replication protein variant standard.