Salicylic acid chemiluminescence immunoassay kit as well as preparation and application thereof

Abstract

The invention belongs to the field of salicylic acid detection, and particularly relates to a salicylic acid chemiluminescence immunoassay kit as well as preparation and application thereof. The kit provided by the invention comprises an R1 reagent (1-3 [mu] m carboxyl magnetic bead coated salicylic acid antibody, a magnetic bead preservation buffer solution containing glycine and bovine serum albumin) and an R2 reagent (alkaline phosphatase coupled salicylic acid synthetic antigen, and an enzyme working solution containing mouse IgG and MgCl2), and by combining a flow injection technology, the detection sensitivity of the kit is improved. The problems that no special kit exists in the existing salicylic acid detection, the high performance liquid chromatography is high in cost, and the spectrophotometric method is poor in sensitivity are effectively solved. The kit provided by the invention can be stably stored for 1 year at 4 DEG C, the deviation between the detection result and the high performance liquid chromatography is less than + / -1%, 60 samples can be detected per hour, the cost is low, the operation is simple and convenient, and the kit is suitable for primary medical institutions and large sample detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of salicylic acid detection, and particularly relates to a salicylic acid chemiluminescence immunoassay kit as well as preparation and application thereof. BACKGROUND

[0002] Salicylic acid is the core metabolite of aspirin (acetylsalicylic acid) in the human body. As a classic non-steroidal drug, aspirin is widely used for antipyretic, analgesic, anti-inflammatory and cardiovascular disease prevention. Due to the significant difference in liver metabolic enzyme activity among patients, the blood concentration of salicylic acid fluctuates greatly. When the concentration is too low (<10 μg / mL), the therapeutic effect is insufficient, and when the concentration is too high (>30 μg / mL), it is easy to cause gastrointestinal ulcers, liver and kidney function damage, salicylic acidemia and other serious adverse reactions. Therefore, it is necessary to accurately monitor the blood concentration of salicylic acid to achieve "dose individualization" in clinical practice, which is a key link to ensure the safety and effectiveness of drug use.

[0003] At present, the technologies for salicylic acid detection at home and abroad mainly focus on two categories. High performance liquid chromatography (HPLC) is the most accurate method for existing detection, which has high accuracy, but the sample pretreatment is complex, the equipment dependence is high, and professional technical personnel are needed for operation and maintenance, the detection cost is high, and only 5-8 samples can be detected per hour by a single equipment, which cannot meet the demand of large sample detection. The operation of spectrophotometry is relatively simple, but the sensitivity is very low, the minimum detection limit is usually >0.5 ng / mL, which cannot detect low concentration salicylic acid; the specificity is poor, and it is easily interfered by endogenous substances such as serum bilirubin, hemoglobin and uric acid, and the detection result deviation is often large; the batch difference is large, and the results are not comparable.

[0004] At present, there is no special immunoassay kit designed for the characteristics of salicylic acid molecules in the field of salicylic acid detection. Therefore, it is necessary to develop a special, stable, accurate and low-cost salicylic acid chemiluminescence immunoassay kit. SUMMARY

[0005] In view of the above technical problems of the prior art, the present application aims to provide a salicylic acid chemiluminescence immunoassay kit, and preparation and application thereof. The kit provided by the present application comprises R1 reagent (1-3 μm carboxyl magnetic beads coated with salicylic acid antibody, and the magnetic bead storage buffer contains glycine and bovine serum albumin) and R2 reagent (alkaline phosphatase coupled salicylic acid synthetic antigen, and the enzyme working solution contains mouse IgG and MgCl2), which is combined with flow injection technology, and effectively solves the problems of no special kit for existing salicylic acid detection, high cost of high performance liquid chromatography and poor sensitivity of spectrophotometry.

[0006] The application discloses a preparation method of a salicylic acid chemiluminescence immunoassay kit, which comprises the following steps: (1) Preparation of R1 reagent: take the carboxyl-modified magnetic beads, resuspend them in 40-60 mM MES buffer, and then magnetically separate and discard the supernatant; add a mixed solution of EDC / NHS, activate for 30 min at 25°C, magnetically separate and discard the activated solution; add the salicylic acid antibody, couple for 4 h at 25°C, magnetically separate and discard the coupling solution; block with a magnetic bead storage buffer for 3 h, dilute to a magnetic bead concentration of 0.1-1 mg / mL, and obtain the R1 reagent; (2) Preparation of R2 reagent: mix the salicylic acid synthetic antigen with ALP, and then dilute to volume with PBS, and dialyze overnight; add glutaraldehyde, and react in the dark for 3 h at 25°C; purify by dialysis with TBS, add an equal volume of glycerol, dilute to a protein concentration of 0.1-1 mg / mL with an enzyme working solution, and obtain the R2 reagent.

[0007] Preferably, in step (1), the mass ratio of EDC / NHS to the carboxyl-modified magnetic beads is 1:(10-50).

[0008] Preferably, in step (1), the particle size of the carboxyl-modified magnetic beads is 1-3 μm.

[0009] Preferably, in step (1) of preparation of the R1 reagent, the mass ratio of the hydroxyl magnetic beads to the olanzapine antibody is 1:(2-20).

[0010] Preferably, in step (1), the mass ratio of the salicylic acid antibody to the carboxyl-modified magnetic beads is 1:(5-20).

[0011] Preferably, in step (1), the mass ratio of the salicylic acid antibody to the carboxyl-modified magnetic beads is 1:(2-20).

[0012] Preferably, in step (1), the magnetic bead storage buffer comprises 50 mM Tris, 0.05-1.0% glycine, 0.1% Tween-20, 0.1% Proclin 300, and 1.0-5.0% bovine serum albumin, with a pH of 7.0.

[0013] Preferably, in step (2), the mass ratio of ALP to the salicylic acid synthetic antigen is (1-10):1.

[0014] Preferably, in step (2), the enzyme working solution comprises 50 mM Tris, 0.1% Tween-20, 0.1% Proclin 300, 0.05-5 μg / mL mouse IgG, and 20-100 μM MgCl2.

[0015] A salicylic acid chemiluminescence immunoassay kit, which is prepared by any one of the above-mentioned methods for preparing a salicylic acid chemiluminescence immunoassay kit.

[0016] The application of the salicylic acid chemiluminescence immunoassay kit is combined with a flow injection technology (FIA) to quantitatively detect salicylic acid in serum, and parameters of the flow injection technology are as follows: a peristaltic pump flow rate is 0.5-5 mL / min, a sample injection volume is 50-100 μL, a reaction coil length is 1-2 m, and a column temperature is 37 DEG C.

[0017] Compared with the prior art, the application has the following beneficial effects: The application provides a salicylic acid chemiluminescence immunoassay kit and preparation and application thereof, and has the following characteristics: (1) The kit is blank filled: the special chemiluminescence immunoassay kit for salicylic acid is developed for the first time, large equipment such as HPLC is not needed, equipment investment cost is reduced, and single detection cost is reduced.

[0018] (2) Stability is significantly improved: R1 reagent is blocked by the cooperation of glycine and BSA, can be stored at 4 DEG C for one year, the activation efficiency of magnetic beads reaches 95%, the recovery rate is greater than 98%, and the CV is less than 1.3%.

[0019] (3) High detection accuracy: mouse IgG in the enzyme working solution can specifically combine with interfering antigens in blood samples, the non-specific combination rate is reduced to 3%; MgCl2 can competitively combine with serum albumin, the final detection result has a deviation of less than ±1% from HPLC, and the minimum detection limit reaches 0.05 ng / mL.

[0020] (4) High detection efficiency: combined with the FIA technology, the single sample detection time is 5 minutes, 60 samples can be processed per hour, and the technology is suitable for batch detection of large samples.

[0021] (5) Simple operation: the sample only needs to be diluted by 1:10, without complex pretreatment, an operator can operate after simple training, and the operation meets the easy operation requirement.

[0022] (6) The detection kit prepared in the application can further reduce the difference between the test results of blood plasma in different anticoagulation blood collection tubes and the test results of serum compared with conventional kits. DETAILED DESCRIPTION

[0023] The following examples are provided to better further understand the application, and do not limit the content and protection scope of the application, and do not constitute a limitation, anyone who obtains any product same or similar to the application under the enlightenment of the application or combines the application with other prior art features falls within the protection scope of the application.

[0024] The specific experimental steps or conditions are not indicated in the examples, and can be performed according to the conventional experimental steps or conditions described in the literature in the field. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by market purchase.

[0025] Example 1: A preparation method of a salicylic acid chemiluminescence immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: 10 mg of carboxyl magnetic beads (particle size 1 μm) were placed in a sterile coating tube, 2.5 mL of activation buffer (50 mM MES, pH 6) was added, vortexed at 2000 rpm for 10 s, then placed on a magnetic stand for magnetic separation for 5 min, and the supernatant was discarded; EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) were weighed and dissolved in the activation buffer to a concentration of 10 mg / mL, mixed at a volume ratio of 1:1, and 200 μL was added to the coating tube (EDC / NHS to magnetic bead mass ratio of 1:20), and 25°C roller shaft oscillation activation for 30 min. The activated solution was discarded by magnetic separation, and the magnetic beads were resuspended with 1 mL of magnetic bead coating buffer (50 mM Tris, pH 7.0, 1.0% glycine, 0.1% Tween-20, 0.1% Proclin 300, 2.0% bovine serum albumin), 200 μg of salicylic acid monoclonal antibody was added, and 25°C roller shaft oscillation coupling was performed for 4 h (mixed every 30 min to ensure uniform coupling). The coupling solution was discarded by magnetic separation, 2.5 mL of magnetic bead storage buffer (containing glycine and BSA) was added, and 25°C roller shaft blocking was performed for 3 h; the blocking solution was discarded by magnetic separation, the magnetic bead storage buffer was diluted to a magnetic bead concentration of 0.5 mg / mL, and was divided into brown reagent bottles, and was stored at 4°C after sealing.

[0026] (2) Preparation of R2 reagent: 100 μg of salicylic acid synthetic antigen (salicylic acid-bovine serum albumin conjugate coupling ratio 15:1) and 500 μg of alkaline phosphatase (ALP, specific activity ≥1000 U / mg) were placed in a 2 mL centrifuge tube, PBS buffer was added to a volume of 150 μL, vortexed and transferred to a 3.5 kDa dialysis bag, and dialyzed overnight with 1 L of 1× PBS buffer (4°C) with stirring. The mixed solution in the dialysis bag was taken out, 10 μL of 1% glutaraldehyde solution was added to obtain a mixed solution with a final concentration of 0.1%, and the solution was reacted at 25°C for 3 h with light protection (mixed gently every 30 min). The reaction solution was transferred to a dialysis bag, dialyzed overnight with 1 L of 1× TBS buffer (4°C) with stirring; the concentration of the conjugate after dialysis was determined by ultraviolet spectrophotometry (wavelength 280 nm), an equal volume of glycerol was added to the dialyzed enzyme label, and the enzyme working solution (50 mM Tris, 0.1% Tween-20, 0.1% Proclin 300, 1.0 μg / mL mouse IgG, and 40 μM MgCl2) was added to dilute the protein concentration to 0.5 mg / mL (the effective concentration of the enzyme label was 1 / 2 of the measured average value), and was divided into light-protected reagent bottles and stored at -15±3°C.

[0027] The application detection method of the kit is as follows: S1 sample pretreatment: take 10 μL of serum sample, dilute with PBS buffer at 1:10; S2 flow injection parameter setting: peristaltic pump flow rate 2 mL / min, six-way injection valve injection volume 75 μL, reaction coil with inner diameter 0.5 mm and length 1 m, column temperature setting 37℃; S3 immune reaction and signal detection: inject diluted sample, R1 reagent (sample:R1 volume ratio 1:1) successively by peristaltic pump, incubate at 37℃ in the reaction coil for 5 min to form "magnetic bead-antibody-salicylic acid" complex; inject R2 reagent (sample:R2 volume ratio 1:1) again, react at 37℃ for 10 min; finally inject chemiluminescent substrate 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl) phenyl-1,2-dioxane, and the detector records the luminescence intensity (RLU); S4 concentration calculation: according to the "concentration-RLU" standard curve drawn by salicylic acid standard (0.1 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL), the actual concentration of salicylic acid in the sample is calculated by linear regression. The detection results are shown in the following table:

[0028] Example 2: The particle size (1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm) of the carboxyl magnetic beads in the preparation step of R1 reagent was screened, and the rest of the steps and experimental conditions were the same as in Example 1. The detection results are shown in the following table:

[0029] From the above detection data, the recovery rate of 1 μm carboxyl magnetic beads at high, medium and low concentrations of 1 ng / mL, 50 ng / mL and 100 ng / mL was all >98%, CV <2.1%, 12 month recovery rate >98.5%, and the antibody coupling amount (8.5 μg / mg) was significantly higher than that of 3 μm magnetic beads (4.8 μg / mg), which confirmed that small particle size magnetic beads could improve the coupling efficiency and long-term stability, and solve the problem of poor stability of existing magnetic beads.

[0030] Example 3: The concentration (40 mM, 45 mM, 50 mM, 55 mM, 60 mM) of MES in the preparation step of R1 reagent was screened, and the rest of the steps and experimental conditions were the same as in Example 1. The detection results are shown in the following table:

[0031] From the above detection data, the magnetic bead activation efficiency at 50 mM MES concentration is 95%, the recovery rate at high, medium and low concentrations is >98%, and the CV is <1.3%, which is significantly better than 40 mM (activation efficiency 82%, CV 3.8%), indicating that this concentration can maximize the magnetic bead activation effect and ensure the uniformity of antibody coupling.

[0032] Example 4: Screening of the mass ratio of EDC / NHS to magnetic beads (1:10, 1:20, 1:30, 1:40, 1:50) in the preparation step of R1 reagent, and the rest of the steps and experimental conditions are the same as in Example 1. The detection results are shown in the table below:

[0033] From the above detection data, the coupling efficiency is 95% when the mass ratio of EDC / NHS to magnetic beads is 1:20, the recovery rate at high, medium and low concentrations is >98%, and the CV is <1.3%, while the coupling efficiency is only 78% when the ratio is 1:50, and the CV is 4.0%; Therefore, the ratio of 1:20 is the optimal parameter for coupling reaction, too much will cause reagent waste, and too little will not be fully coupled.

[0034] Example 5: Screening of the mass percentage of glycine (0, 0.05%, 0.15%, 0.65%, 1.0%) and bovine serum albumin (0, 1.0%, 2.0%, 3.5%, 5.5%) in the magnetic bead coating buffer in the preparation step of R1 reagent, and the rest of the steps and experimental conditions are the same as in Example 1. The detection results are shown in the table below:

[0035] From the above detection data, the recovery rate is still >98% after 12 months with 0.65% glycine + 5.5% BSA combination, and the CV is <2%, and the non-specific binding rate is 3.2%, which is much better than single component (such as 1.0% glycine + 0% BSA, the recovery rate is 85%, and the CV is 10.6%), which proves that the synergistic blocking effect of glycine and BSA can greatly extend the shelf life of the reagent and prolong the storage time of the reagent.

[0036] Example 6: Screening of the concentrations of mouse IgG (0, 0.05 μg / mL, 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL) and MgCl2 (0, 20 μM, 40 μM, 70 μM, 100 μM) in the enzyme working solution in the preparation step of R2 reagent, and the rest of the steps and experimental conditions are the same as in Example 1. The detection results are shown in the table below:

[0037] From the above detection data, it can be seen that the combination of 2.5 μg / mL mouse IgG and 70 μM MgCl2 has a deviation of <±1% at three concentrations of high, medium and low, and a non-specific binding rate of 3.0%, while the deviation is >±5% when any component is missing, which confirms that the combination can eliminate the interference of blood sample matrix and improve the detection accuracy.

[0038] Example 7: In the preparation step of R2 reagent, ALP-antigen coupling is carried out by SMCC method, and the remaining steps and experimental conditions are the same as those in Example 1. The detection results are shown in the following table:

[0039] From the above detection data, it can be seen that the coupling efficiency of glutaraldehyde method is 85%, the enzyme activity retention rate is 90%, and the CV of high, medium and low concentrations is <1.3%, which is significantly better than that of SMCC method (coupling efficiency 78%, CV 3.5%), so it can be determined that the glutaraldehyde method is the optimal coupling scheme, which can guarantee the activity of enzyme-labeled antigen.

[0040] Example 8: The flow rate (1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min) of peristaltic pump, the injection volume (50 μL, 60 μL, 70 μL, 80 μL, 100 μL) and the length of reaction coil (0.5 m, 1 m, 1.3 m, 1.6 m, 2 m) in the application detection method of the kit are screened, and the remaining steps and experimental conditions are the same as those in Example 1. The detection results are shown in the following table:

[0041] From the above detection data, it can be seen that when the flow rate of peristaltic pump in flow injection technology is 2 mL / min, the injection volume is 60 μL, and the coil is 1 m, the single sample detection time is 5 min, the CV of high, medium and low concentrations is <1.3%, 65 samples can be detected per hour, which takes into account the efficiency and accuracy, which is better than high flow rate (CV 4.5% when 5 mL / min) or short coil (CV 3.5% when 0.5 m), which solves the problem of low efficiency and large error of manual operation.

[0042] Comparative Example 1: Amino magnetic beads are used instead of hydroxyl magnetic beads, and the remaining steps and experimental conditions are the same as those in Example 1. The detection results are shown in the following table:

[0043] From the above detection data, it can be seen that the recovery rate of carboxyl magnetic beads is >97.5% in 6 months, and the antibody coupling amount is 8.5 μg / mg, while the recovery rate of amino magnetic beads is only 82%, and the coupling amount is 5.2 μg / mg, which confirms that the selection of carboxyl magnetic beads in the present application can improve the stability of reagent and detection sensitivity.

[0044] Comparative Example 2: The flow injection technology was used in the kit detection, and the conventional manual operation was used instead, and the remaining steps and experimental conditions were the same as those of Example 1. The detection results are shown in the following table:

[0045] From the above detection data, it can be seen that the FIA automatic operation can detect 65 samples per hour, and the CV is less than 2.1%; the manual operation can only detect 15 samples per hour, and the CV is greater than 4.1%. The efficiency is improved by 4.3 times, and the error is reduced by 60% after the combination of the FIA technology, which can well meet the detection needs of large samples.

[0046] Comparative Example 3: The existing HPLC method was used for detection, and the chromatographic conditions were as follows: C18 column (250 mm x 4.6 mm), mobile phase methanol-0.1% phosphoric acid (30:70), flow rate 1.0 mL / min, detection wavelength 230 nm; sample pretreatment: 100 μL serum, 200 μL acetonitrile to precipitate protein, 12000 rpm centrifugation for 15 min, and the supernatant was injected. The detection results are shown in the following table:

[0047] From the above detection data, it can be seen that the detection time (5 min) of the present application is only 1 / 8 of that of HPLC, and the cost (10 yuan / time) is 1 / 8 of that of HPLC, and the recovery rate (98.0%-100.5%) is not much different from that of HPLC (99.0%-99.8%), which greatly reduces the detection cost.

[0048] Comparative Example 4: The existing spectrophotometric method was used for detection, and the detection wavelength was 296 nm; sample pretreatment: 500 μL serum, 100 μL trichloroacetic acid (10%) to precipitate protein, 8000 rpm centrifugation for 10 min, and the supernatant was detected. The detection results are shown in the following table:

[0049] From the above detection data, it can be seen that the minimum detection limit (0.05 ng / mL) of the present application is 1 / 10 of that of the spectrophotometric method (0.5 ng / mL), the non-specific interference rate (3.2%) is 1 / 5 of that of the spectrophotometric method (15.2%), and the CV of high, medium and low concentrations is less than 2.1%. The method provided by the present application is better than the spectrophotometric method (CV 3.7%-6.2%), and can meet the needs of accurate detection.

[0050] Comparative Example 5: The same sample was detected by using the conventional kit and the kit prepared by the present application, and the detection results are shown in the following table:

[0051] From the detection data in the above table, it can be known that the prepared detection kit can further reduce the difference between the test results of different anticoagulation blood collection tubes and the test results of serum compared with the conventional kit.

[0052] In summary, by means of targeted parameter optimization and technical organic coordination, the salicylic acid chemiluminescence immunoassay detection kit effectively solves the problems of no special kit, poor stability, low accuracy, low efficiency, high cost and short storage time in the prior art.

[0053] Obviously, the above embodiments are only examples for clearly illustrating the present application, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing a salicylic acid chemiluminescent immunoassay kit, characterized in that, Includes the following steps: (1) Preparation of R1 reagent: Take carboxyl-modified magnetic beads, resuspend them in 40~60mM MES buffer, and then magnetically separate and discard the supernatant; add EDC / NHS mixture, activate at 25℃ for 30min, and magnetically separate and discard the activation solution; add salicylic acid antibody, couple at 25℃ for 4h, and magnetically separate and discard the coupling solution; Block with magnetic bead preservation buffer for 3 hours, then dilute to a magnetic bead concentration of 0.1~1 mg / mL to obtain reagent R1; (2) Preparation of R2 reagent: The salicylic acid synthesized antigen was mixed with ALP, and dialyzed overnight after being brought to a final volume with PBS; glutaraldehyde was added and reacted at 25°C in the dark for 3 hours; the mixture was purified by dialyzed with TBS, and an equal volume of glycerol was added. The mixture was then diluted with enzyme working solution to a protein concentration of 0.1~1 mg / mL to obtain R2 reagent.

2. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1), the mass ratio of EDC / NHS to carboxyl-modified magnetic beads is 1:(10-50).

3. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1), the particle size of the carboxyl-modified magnetic beads is 1~3μm.

4. The preparation method of the salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1), the mass ratio of the salicylic acid antibody to the carboxyl-modified magnetic beads is 1:(5~20).

5. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1), the mass ratio of the salicylic acid antibody to the carboxyl-modified magnetic beads is 1:(2~20).

6. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1), the magnetic bead preservation buffer contains 50 mM Tris at pH 7.0, 0.05-1.0% glycine, 0.1% Tween-20, 0.1% Proclin 300, and 1.0-5.0% bovine serum albumin.

7. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (2), the mass ratio of ALP to salicylic acid to synthesize antigen is (1~10):

1.

8. The method for preparing a salicylic acid chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (2), the enzyme working solution contains 50 mM Tris, 0.1% Tween-20, 0.1% Proclin 300, 0.05~5 μg / mL mouse IgG and 20~100 μM MgCl2.

9. A salicylic acid chemiluminescent immunoassay kit prepared according to any one of claims 1 to 8.

10. The application of the salicylic acid chemiluminescent immunoassay kit as described in claim 9, characterized in that, The kit is combined with flow injection technology for the quantitative detection of salicylic acid in serum. The parameters of the flow injection technology are: peristaltic pump flow rate 0.5~5mL / min, injection volume 50~100μL, reaction coil length 1~2m, and column temperature 37℃.

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