CN107219357A - A kind of ELISA kit for detecting methotrexate (MTX) and its application - Google Patents

Abstract

The present invention provides an ELISA kit for detecting methotrexate and its application. The kit includes the following reagents: 96-well microwell plate coated with methotrexate, methotrexate monoclonal antibody, antibody Diluent, HRP-labeled goat anti-mouse secondary antibody, methotrexate standard, wash solution concentrate, sample diluent, TMB chromogenic solution and stop solution. The present invention obtains a stable, fast and simple methotrexate detection kit through the combination of various reagents and the improvement of reagent parameters, and the standard deviation value of the kit is small when applied. It can be used for the blood drug concentration detection of patients who use large doses of methotrexate and the detection of drug concentrations in serum of patients with low doses of methotrexate treatment.

Description

A kind of ELISA kit for detecting methotrexate and its application

technical field

The invention belongs to the field of immune detection, and in particular relates to an ELISA kit for detecting methotrexate and its application.

Background technique

Methotrexate (MTX) is a drug used to treat cancer and autoimmune diseases. It is designed as an antifolate to inhibit the metabolism of folic acid. In cancer therapy, methotrexate competitively inhibits dihydrofolate reductase (DHFR), which converts dihydrofolate to active tetrahydrofolate, by blocking folate binding. Inhibition of DHFR results in inhibition of purine and pyrimidine base synthesis, effectively limiting DNA and RNA synthesis and cancer cell growth. In autoimmune diseases, especially in the treatment of rheumatoid arthritis, methotrexate appears to affect several pathways leading to inhibition of T cell activation. These effects include inhibition of T cell expression of intercellular adhesion molecules, inhibition of methyltransferase activity and increased CD95 sensitivity, leading to apoptosis of activated T cells.

Monitoring methotrexate levels is important to ensure appropriate levels during or during treatment. High levels of methotrexate can lead to toxicity and potential renal failure as well as immunosuppression. In addition, methotrexate is known to interact with a wide variety of medications, leading to additional complications. Determining the presence of methotrexate in samples from subjects can help interpret study results. Methotrexate is established as one of the most effective and safe treatments for rheumatoid arthritis. The safety analysis ensures that methotrexate will continue to be used in combination with other new or established drugs in new studies. The same is true when it is used as a cancer therapeutic.

Large doses of MTX are often used in the treatment of tumors and leukemia. The latest diagnosis and treatment plan for children with acute lymphoblastic leukemia proposes that the dosage of methotrexate is 2-5g/(m2.24h), and the serum concentration of patients can be as high as 20μmol/L. The methods mainly include high performance liquid chromatography, fluorescence polarization immunoassay, and enzyme amplification immunoassay. In recent years, MTX has been used in the treatment of systemic autoimmune diseases such as rheumatoid arthritis, Sjogren's syndrome, and psoriatic arthritis. L, the concentration difference between the two is nearly 1000 times. Traditional detection methods are difficult to detect, and high-performance liquid chromatography-mass spectrometry is now commonly used for detection. However, the sample pretreatment of chromatography-mass spectrometry is complicated, and the operation steps are relatively cumbersome, and the cost of chromatography-mass spectrometer is high, and the detection cost is high, so it is not suitable for large sample detection and grass-roots development.

Contents of the invention

In view of the above-mentioned defects in the prior art, the main purpose of the present invention is to provide a kind of ELISA kit and application thereof for detecting methotrexate, said kit can quickly, easily and stably detect methotrexate in samples , and the standard deviation is small.

In order to achieve the above object, the present invention adopts the following technical scheme: an ELISA kit for detecting methotrexate, said kit comprising the following reagents: a 96-well microplate coated with methotrexate, methotrexate mono Cloned antibody, antibody diluent, HRP-labeled goat anti-mouse secondary antibody, methotrexate standard, wash solution concentrate, sample diluent, TMB chromogenic solution and stop solution.

As a further preference, the preparation method of the 96-well microplate coated with methotrexate comprises the following steps:

Dilute the coating material with coating buffer, add the diluted coating material into the microwells, seal with the plate sealing film, coat overnight or incubate for 2-4 hours in an incubator;

After coating overnight or after incubation, shake off the liquid in the plate and pat dry, wash the plate, pat dry, add blocking solution to each well, seal with plate sealing film, and incubate in an incubator for 1-3 hours to seal;

After the sealing, take the plate and pat it dry, add the soaking liquid, and incubate in the thermostat for 1-3h to soak the plate;

After the soaking of the board, the board was taken out and patted dry, and the board was evacuated.

As a further preference, the coating agent includes methotrexate-egg albumin.

As a further preference, the coating buffer includes CB buffer; the washing solution for washing the plate includes 1×TBST solution, the blocking solution includes 10×PBS solution, bovine serum and bovine serum albumin, and the immersion The plate solution includes 10×PBS solution, sucrose and trehalose.

As a further preference, the temperature of the overnight coating is 4°C, and the temperature of the incubator is 37°C.

As a further preference, the methotrexate monoclonal antibody is secreted and produced by hybridoma cells with a deposit number of CCTCC NO: C201726.

As a further preference, the antibody diluent includes 10×PBS solution, 2% bovine serum albumin and 10% bovine serum.

As a further preference, the sample diluent includes PBS solution and bromophenol red indicator.

As a further preference, the stop solution includes 2-3 mol/L H ₂ SO ₄ solution.

As a further preference, the concentrated lotion solution is a 10×TBST solution.

An application of an ELISA kit for detecting methotrexate, characterized in that: the kit is used for the blood drug concentration detection of patients using large doses of methotrexate and the detection of drugs in serum of patients with low doses of methotrexate treatment Concentration detection.

The beneficial effects of the present invention are:

(1) The present invention obtains a stable, rapid and simple methotrexate detection kit through the combination of various reagents and the improvement of reagent parameters, and the standard deviation value of the kit is relatively small during application.

(2) When the test kit of the present invention is used to detect methotrexate in a sample, the sample pretreatment is simpler than chromatography mass spectrometry, and the operation steps are simplified, and the test kit of the present invention does not need to use expensive instruments, the detection cost is low, and it is suitable for large samples Inspection and grassroots development.

(3) The specificity of the assay was determined by serially diluting potential cross-reactants in the assay buffer of the kit of the invention and running them in the assay. The results show that metabolites of methotrexate, folic acid analogues, other analogues and other substances similar in structure to methotrexate are quantitatively detected in the analysis; the specificity of the kit of the invention is high.

(4) The kit of the present invention has high sensitivity and accuracy, good anti-interference performance, and does not cross-react with the main metabolite 7-hydroxymethotrexate during the determination of methotrexate.

(5) The kit of the present invention can be used simultaneously for the detection of blood drug concentration of MTX during high-dose MTX chemotherapy and low-dose treatment of autoimmune diseases such as rheumatoid arthritis.

Description of drawings

Fig. 1 is a schematic diagram of the standard curve fitting of the ELISA kit for detecting methotrexate in Example 1 of the present invention.

detailed description

The present invention provides an ELISA kit for detecting methotrexate and its application. The kit can quickly, easily and stably detect methotrexate in a sample, and the standard deviation value is small.

Existing detection methods such as high-performance liquid chromatography can separate drugs from metabolites and endogenous substances, and have strong specificity. The longer measurement time is not suitable for the requirements of simple operation, high degree of automation, and short measurement cycle required by clinical testing. For example, the immunoassay is fast and easy to operate, but its results are easily affected by cross-reactions. In the process of detecting methotrexate, the current kits often have the disadvantages of obvious cross-reactions and long detection time. Therefore, it is of great significance to establish a stable, fast, simple and low-cost MTX detection method.

ELISA is a simple, sensitive and specific detection method that can be used to detect various blood drug concentrations. The indirect competitive ELISA method can detect the plasma concentration of MTX in the order of μg/mL, which can be used to detect the plasma concentration of patients who use large doses of MTX. The concentration of MTX in the serum of autoimmune disease patients with low-dose treatment is on the order of ng/mL, and the traditional ELISA detection method cannot detect the drug concentration in the serum of patients with low-dose treatment of MTX. Therefore, it is necessary to develop a blood drug concentration that can be commonly used in high-dose MTX chemotherapy and low-dose treatment of autoimmune diseases such as rheumatoid arthritis.

In order to solve the above defects, the main ideas of the embodiments of the present invention are:

The ELISA kit for detecting methotrexate in the embodiment of the present invention includes the following reagents: 96-well microwell plate coated with methotrexate, methotrexate monoclonal antibody, antibody diluent, HRP-labeled goat anti-mouse secondary antibody , standard methotrexate, wash solution concentrate, sample diluent, TMB chromogenic solution and stop solution.

The preparation method of the 96-well microwell plate coated with methotrexate is as follows: a. Coating: use the coating buffer solution to dilute the coating material to a suitable concentration, add it to the microwell, 100ul per well, to seal Seal the plate with film, coat overnight at 4°C or incubate at 37°C in an incubator for 2 hours; b. Seal: after overnight coating or coating at 37°C, shake off the liquid in the plate, pat dry, wash the plate twice, The washing solution is 1×TBST, 250ul per well, and the washing time is 30s. After washing the plate, pat it dry, add 200ul of blocking solution to each well, seal it with a sealing film, incubate in a 37°C incubator for 1.5h, set the temperature at 37°C; c. soak the plate: after sealing, take the plate and pat dry, Add 200ul plate soaking solution and incubate in a 37°C incubator for 2h; d. Standby: take the plate and pat it dry, vacuumize the plate and place it at 4°C for standby.

The kit can be used for the detection of blood drug concentration of patients using large doses of MTX and the detection of drug concentrations in blood serum of patients with low doses of MTX treatment.

In order to make the above and other objects, features, and advantages of the present invention more comprehensible, the following examples are given to illustrate the ELISA kit for detecting methotrexate of the present invention and its application.

Example 1

Example 1 of the present invention detects the ELISA kit for methotrexate, including the following reagents: 96-well microwell plate coated with methotrexate, methotrexate monoclonal antibody, antibody diluent, HRP-labeled goat anti-mouse II Antibody, Methotrexate Standard, Washing Solution Concentrate, Sample Diluent, TMB Chromogenic Solution and Stop Solution.

1. The preparation of each reagent is as follows:

a. Coating CB buffer: pH9.6 carbonate buffer:

b.25×PBS:

When used, dilute 25×PBS 25 times to obtain 1×PBS.

c.10×TBST:

When used, dilute 10×TBST 10 times to obtain 1×TBST.

d. Blocking solution:

e. Dipping solution:

f. Antibody diluent:

g. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

h. Calibrator diluent and sample diluent: 1×PBS+0.0025% bromophenol red indicator.

I: The stop solution is 2mol/L H ₂ SO ₄ solution.

J: The lotion concentrate is 10 x TBST.

K: Substrate color development solution A: sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water to 500ml; substrate color development solution B: disodium edetate 0.2g, citric acid 0.95 g, glycerol 50ml, take 0.15g TMB and dissolve in 3ml DMSO, add distilled water to 500ml. When using, mix 1:1, and then add a certain amount to each well.

L: The methotrexate monoclonal antibody is secreted by the hybridoma cell with the deposit number CCTCC NO: C201726. The depository unit is: China Center for Typical Culture Collection (Wuhan University), the name of the culture is: hybridoma cell line 4F2-1A9, the deposit number is: CCTCC NO: C201726, and the deposit date is: 2017.4.11.

M: standard methotrexate (National Institute for the Control of Pharmaceutical and Biological Products, batch number: 00138-200603) was diluted to concentrations of 0.025, 0.05, 0.1, 0.2, 0.5, and 1 μmol/L as required.

N: HRP-labeled goat anti-mouse secondary antibody, prepared by the applicant company.

2. Sample processing and use

a. Human plasma sample: Dilute the sample to 20 times (when the content of methotrexate in the sample is relatively low) or 40 times (when the content of methotrexate in the sample is relatively high) with sample diluent;

b. Human serum sample: dilute the serum sample to 160 times with sample diluent;

c. Treat the sample with the corresponding method, add 30ul of the corresponding standard product or 30ul of the treated sample to each well, then add 70ul of the antibody working solution, shake and mix, and incubate in a 37°C oven for 1h;

3. Coating with methotrexate-OVA (egg albumin) conjugate

a. Coating: Use methotrexate-OVA as the coating source, dilute the coating source to 0.4ug/ml, 0.2ug/ml and 0.1ug/ml with CB buffer solution, react overnight at 4°C or at 37°C 2h, process the board according to the method described above;

b. Antigen-antibody specific reaction: Dilute the methotrexate antibody to 0.5mg/ml with antibody diluent, then dilute the antibody solution from 1:1000, and add it to the corresponding coated well, 100ul per well . Place at 37°C and incubate for 1 hour, shake off the liquid in the plate, wash the plate 3 times with 1×TBST, and pat dry;

c. Add secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody 5000 times with antibody diluent, mix well, add 100ul to each well, incubate at 37°C for 40min, wash the plate 3 times with TBST, and pat dry

d. Color development: Add 100ul of substrate to each well, react at 37°C for 20min

e. Stop the reaction: add 0.3mol/l H2SO450ul to each well, and immediately measure the absorbance value OD450 at 450nm with a microplate reader

f. Interpretation of results: The antibody concentration corresponding to the lowest coating concentration used when the OD450 value is close to 2.0 is the titer of the antibody

4. The concentration confirmation method of the antibody is used, and the dilution ratio of the primary antibody is explored by the chessboard method.

(1) Antigen coating: Dilute antigen to 2ug/ml with coating solution, add 100 μL/well into polystyrene 96-well reaction plate, and place overnight at 4°C.

(2) Washing: discard the liquid in the hole the next day, and wash with the washing liquid 3 times.

(3) Blocking: add 150 μL/well blocking solution, and place at room temperature for 0.5 h.

(4) Washing: Wash 3 times with washing liquid.

(5) Add primary antibody: add antiserum (centrifuge at 4000r/min for 10min to obtain supernatant after taking blood at 4°C overnight), dilute the serum at 1:200, 1:400, 1:800, 1:1600, 1 :3200, 1:6400, 1:12800... for doubling dilution (blank serum as negative control), 100ul per hole, incubate for 1h.

(6) Washing: wash 3 times with washing liquid.

(7) Add enzyme-labeled anti-antibody: add HRP-labeled goat anti-rabbit IgG (1:5000, diluted with enzyme), 100 μl/well, and incubate at 37°C for 40 minutes.

(8) Washing: Wash 5 times with washing solution and 2 times with distilled water.

(9) Color development: Add 100 μL/well of freshly prepared substrate solution, and place in dark place at room temperature for 5-30 minutes

(10) Stop reaction, colorimetry: add 50 μL/well stop solution. The color turns yellow; use a microplate reader to measure the absorbance of each well at 450nm;

5. Operation steps of methotrexate ELISA kit

5.1 Move various reagents to room temperature (20-25°C) to equilibrate for at least 30 minutes, prepare relevant reagents according to the instructions, and set aside.

5.2 Take out the microwell strips and plate racks as needed, put the unused microwell strips back into the aluminum foil bag, and store them at 2-8°C.

5.3 Add 30 μL of standard/mixed sample to each well. It is recommended to make duplicate wells for the standard/sample, then add 70 μL of antibody, seal the plate with plate stickers, shake and mix gently, and react at 37°C for 1 hour.

5.4 Take out the microporous plate, shake off the liquid in the well, and wash the plate 4 times with the diluted lotion working solution. Soak for 30 seconds each time, 250 μL/well, and pat dry on absorbent paper.

5.5 Add 100 μL of enzyme conjugate to each well, seal the plate with a plate, and react at 37°C for 40 minutes.

5.6 Take out the microporous plate, shake off the liquid in the well, and wash the plate 4 times with the diluted lotion working solution. Soak for 30 seconds each time, 250 μL/well, and pat dry on absorbent paper.

5.7 Color development: Add 100 μL of substrate solution to each well, shake gently, and develop color at 37°C in the dark for 20 minutes.

5.8 Sequentially add 50 μL of stop solution to each well to stop the reaction (the blue color turns yellow immediately). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time is up.

5.9 Use a microplate reader to sequentially measure the optical density (OD value) of each well at a wavelength of 450 nm. Perform detection within 5 minutes after adding the stop solution (it is recommended to use dual-wavelength 450/630nm detection, and read the data within 5 minutes).

Example 2

Aspects 3-5 are similar to Embodiment 1, the difference is:

1. Reagent preparation

a. Coating buffer: same as Example 1

b. 25×PBS: Same as Example 1, when used, dilute 25×PBS 25 times to obtain 1×PBS

c.10×TBST: Same as Example 1, when in use, 10×TBST is diluted 10 times to obtain 1×TBST

d. Blocking solution:

e. Antibody diluent:

f. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

g. Calibrator diluent and sample diluent: 1×PBS++0.0025% bromophenol red indicator

2. Preparation of MTX-OVA-coated microwell plates: a. Coating: Dilute the coated original methotrexate-OVA to a suitable concentration with CB buffer solution, add it to the microwells, 100ul per well, Seal with plate sealing film, coat overnight at 4°C or incubate at 37°C in an incubator for 2 hours; b. Seal: after overnight coating or coating at 37°C, shake off the liquid in the plate, pat dry, wash the plate twice The first time, the washing solution is 1×TBST, 250ul per well, and the washing time is 30s. After washing the plate, pat it dry, add 200ul of blocking solution to each well, seal it with a plate sealing film, incubate in a 37°C incubator for 1.5h, set the temperature at 37°C; Vacuum, store at 4°C for later use

Example 3

Aspects 3-5 are similar to Embodiment 1, the difference is:

1. Reagent preparation

a. Coating buffer: same as Example 1

b. 25×PBS: Same as Example 1, when used, dilute 25×PBS 25 times to obtain 1×PBS

c.10×TBST: Same as Example 1, when in use, 10×TBST is diluted 10 times to obtain 1×TBST

d. Blocking solution:

e. Antibody diluent: 1×PBS+2%BSA+10% bovine serum+0.0025% bromophenol red indicator

f. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

g. Calibrator diluent and sample diluent: 1×PBS++0.0025% bromophenol red indicator

2. Preparation of MTX-OVA-coated microwell plates: a. Coating: Dilute the coated original methotrexate-OVA to a suitable concentration with CB buffer solution, add it to the microwells, 100ul per well, Seal with plate sealing film, coat overnight at 4°C or incubate at 37°C in an incubator for 2 hours; b. Seal: after overnight coating or coating at 37°C, shake off the liquid in the plate, pat dry, wash the plate twice The first time, the washing solution is 1×TBST, 250ul per well, and the washing time is 30s. After washing the plate, pat it dry, add 200ul of blocking solution to each well, seal it with a plate sealing film, incubate in a 37°C incubator for 1.5h, set the temperature at 37°C; Vacuum, store at 4°C for later use

In order to obtain the effect of the anti-methotrexate monoclonal antibody, the following series of tests were carried out on the kit obtained in Example 1 to demonstrate:

Test 1:

The standard products with methotrexate concentrations of 0.025, 0.05, 0.1, 0.2, 0.5, and 1 μmol/L were detected using the microtiter plates coated in Examples 1, 2, and 3, respectively. The detection method is the same as the operation steps of the above-mentioned methotrexate ELISA kit, with the logarithm of each concentration of methotrexate as the abscissa, and the OD value corresponding to each concentration of methotrexate as the ordinate, draw a standard curve to obtain the detection The CV values of the standard curve are shown in Table 1-3.

Table 1: The parameters of the standard curve for the preparation of the methotrexate kit in Example 1

Table 2: The parameters of the standard curve for the preparation of the methotrexate kit in Example 2

Table 3: The parameters of the standard curve for the preparation of the methotrexate kit in Example 3

Conc OD1 OD2 mean SD CV% BLK 0 2.2863 2.1101 2.1982 0.1246 5.67 STD1 0.025 1.5109 1.4055 1.4582 0.0745 5.11 STD2 0.05 1.4456 1.3487 1.3971 0.0685 4.90 STD3 0.1 0.9572 1.1698 1.0635 0.1503 14.14 STD4 0.2 0.4157 0.5369 0.4763 0.0857 17.99 STD5 0.5 0.2145 0.2421 0.2283 0.0195 8.55 STD6 1 0.2336 0.259 0.2463 0.0180 7.29

It can be seen that the kit prepared in Example 1 is better in standard deviation value.

Test 2: Kit specificity test

Assay specificity was determined by serially diluting potential cross-reactants in kit assay buffer and running them in the assay. Metabolites of methotrexate, folate analogs, and other analogs that are structurally similar to methotrexate were quantitatively detected in the assay. Among them, folic acid, 7-hydroxymethotrexate, and DAMPA were detected in the experiment. The results were fitted to a 4 parameter logarithmic equation and the ED50 was determined for each cross-reactant. Divide each ED50 by the ED50 of methotrexate and multiply by 100 to provide percent cross-reactivity.

cross reactant cross reactivity rate Dihydrofolic Acid 0.11% 7-hydroxymethotrexate 0.19% DAMPA 15.3%

Experiment 3: Recycling experiment

Precision (analytical recovery) was calculated by the amount of methotrexate drug spiked at a fixed concentration in methotrexate-free negative sera. A certain amount of high-purity stock solution concentration was added to methotrexate-free negative serum, and the concentration of the drug was determined by analyzing the dose range. Each sample was tested 6 times, and the results were averaged and compared with the original dose. The recovery experiment is to add a fixed amount of the substance to be tested in the blank serum, and then use the kit to test the substance. The ratio between the measured amount and the measured amount is multiplied by 100%, which is the recovery rate.

Test 4: Sensitivity

This sensitivity was determined by interpolation from the mean of 9 separate standard curve runs with replicate data points for each concentration. Sensitivity was determined at 2 standard deviations below the mean net OD of 18 zero standard replicates (2 per standard curve). The detection sensitivity or limit of the assay was 0.25 ppb. The linear range of the kit: 0.25-10ng/ml. The following formula is the four-parameter fitting equation and correlation coefficient of the standard curve.

R2 0.99565

Test 5: Within-assay precision

Intra-assay precision was obtained by 20 single-repeat tests of quality controls at two concentrations of methotrexate

Test 6: Inter-assay precision

The inter-assay precision was determined by testing two concentrations of the methotrexate quality control multiple times over several days (N=10)

Test 7: Methodological Correlation Analysis:

When the kit of the embodiment of the present invention is compared with the TDx methotrexate kit of Abbott, when the methotrexate assay is designed to range from 0.040 μmol/L to 1.000 μmol/L of samples, it has a slope of 1.00 ± 0.10, The correlation coefficient (r) ≥ 0.95, when compared with the liquid chromatography-tandem mass spectrometry (LC/MS/MS) method, for sample concentrations ranging from 0.040 μmol/L to 1.500 μmol/L.

The Passing-Bablok regression method was used to compare the methotrexate ELISA assay with the TDx methotrexate and LC/MS/MS method when conducting studies based on the guidance CLSI document EP9-A329 using serum samples. The data are summarized in the table below, including the data measurement intervals requiring dilutions from studies with the above specimens.

Table 4 ELISA methotrexate vs TDx methotrexate (N=90)

CI confidence interval

Table 5 ELISA methotrexate vs LC/MS/MS (N=100)

Table 6 ELISA methotrexate vs TDx methotrexate (N=120)

Test 8: Anti-interference:

Conduct study EP7-A2.28 Evaluation of Potentially Interfering Drugs as directed by CLSI document to determine if methotrexate concentrations are affected when administered. The drug standards listed below were spiked and sample points of methotrexate concentrations were approximately 0.050 μmol/L and 1.000 μmol/L. Sample assays spiked with methotrexate standards compared to reference samples. The methotrexate assay does not cross-react with the major metabolite 7-hydroxymethotrexate. Unless otherwise stated, the added concentration of the test drug was 1000 μmol/L.

Table 7

Experiment 9: Practical testing of samples

Some patients on methotrexate were selected for actual serum and plasma testing, and the results are as follows.

The above-mentioned technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

(1) The present invention obtains a stable, rapid and simple methotrexate detection kit through the combination of various reagents and the improvement of reagent parameters, and the standard deviation value of the kit is relatively small during application.

(2) When the test kit of the present invention is used to detect methotrexate in a sample, the sample pretreatment is simpler than chromatography mass spectrometry, and the operation steps are simplified, and the test kit of the present invention does not need to use expensive instruments, the detection cost is low, and it is suitable for large samples Inspection and grassroots development.

(3) The specificity of the assay was determined by serially diluting potential cross-reactants in the assay buffer of the kit of the invention and running them in the assay. The results show that metabolites of methotrexate, folic acid analogues, other analogues and other substances similar in structure to methotrexate are quantitatively detected in the analysis; the specificity of the kit of the invention is high.

(4) The kit of the present invention has high sensitivity and accuracy, good anti-interference performance, and does not cross-react with the main metabolite 7-hydroxymethotrexate during the determination of methotrexate.

(5) The kit of the present invention can be used simultaneously for the detection of blood drug concentration of MTX during high-dose MTX chemotherapy and low-dose treatment of autoimmune diseases such as rheumatoid arthritis.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (10)

1. An ELISA kit for detecting methotrexate, characterized in that: said kit includes the following reagents: a 96-well microplate coated with methotrexate, methotrexate monoclonal antibody, antibody diluent , HRP-labeled goat anti-mouse secondary antibody, methotrexate standard, wash solution concentrate, sample diluent, TMB chromogenic solution and stop solution. 2. the ELISA kit of detecting methotrexate according to claim 1, is characterized in that: the preparation method of the 96-well microwell plate coated with methotrexate comprises the steps: Dilute the coating material with coating buffer, add the diluted coating material into the microwells, seal with the plate sealing film, coat overnight or incubate for 2-4 hours in an incubator; After coating overnight or after incubation, shake off the liquid in the plate and pat dry, wash the plate, pat dry, add blocking solution to each well, seal with plate sealing film, and incubate in an incubator for 1-3 hours to seal; After the sealing, take the plate and pat it dry, add the soaking liquid, and incubate in the thermostat for 1-3h to soak the plate; After the soaking of the board, the board was taken out and patted dry, and the board was evacuated. 3. The ELISA kit for detecting methotrexate according to claim 2, characterized in that: said coating source comprises methotrexate-egg albumin. 4. The ELISA kit for detecting methotrexate according to claim 2, characterized in that: the coating buffer comprises CB buffer; the washing solution of the plate washing comprises 1 × TBST solution, and the blocking The liquid includes 10×PBS solution, bovine serum and bovine serum albumin, and the dipping solution includes 10×PBS solution, sucrose and trehalose. 5 . The ELISA kit for detecting methotrexate according to claim 1 , characterized in that: the methotrexate monoclonal antibody is secreted and produced by hybridoma cells whose deposit number is CCTCC NO: C201726. 6 . The ELISA kit for detecting methotrexate according to claim 1 , wherein the antibody diluent comprises 10×PBS solution, 2% bovine serum albumin and 10% bovine serum. 7. The ELISA kit for detecting methotrexate according to claim 1, characterized in that: said sample diluent comprises PBS solution and bromophenol red indicator. 8 . The ELISA kit for detecting methotrexate according to claim 1 , wherein the stop solution comprises 2-3 mol/L H ₂ SO ₄ solution. 9. The ELISA kit for detecting methotrexate according to claim 1, characterized in that: the lotion concentrate is a 10×TBST solution. 10. detect the application of the ELISA test kit of methotrexate as described in any one of claim 1-9, it is characterized in that: described test kit is used for the blood drug concentration detection of large dose methotrexate use patient and small Drug concentration detection in serum of patients with therapeutic dose of methotrexate.