Canine early pregnancy RLN detection kit and detection method thereof

Abstract

The invention relates to a dog early pregnancy RLN detection kit and a detection method thereof. The kit comprises a magnetic bead working solution, an enzyme working solution, a cleaning solution, a chemiluminescent substrate, a calibration product and a quality control product. Wherein the magnetic bead working solution contains toluenesulfonyl magnetic beads labeled by canine RLN monoclonal antibodies, and the enzyme working solution contains canine RLN monoclonal antibodies labeled by alkaline phosphatase and an antibody blocking agent. When the canine early pregnancy RLN detection kit provided by the invention is used for determining the canine RLN concentration in serum, the RLN antibody on the magnetic bead and the RLN antibody on the enzyme marker are simultaneously and rapidly combined with different sites on the RLN antigen to form an antibody-antigen-antibody structure, the combination is more sufficient, the detection sensitivity is high, the antibody blocking agent shortens the window period of detection, and the detection time is shortened. The false positive of the detection result is reduced, and the detection accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic detection, and specifically relates to a canine early pregnancy RLN detection kit and a detection method thereof. Background Art

[0002] Canine early pregnancy hormone (RLN) is also known as canine relaxin. RLN is a polypeptide hormone mainly derived from the corpus luteum of pregnancy, and its molecular structure is similar to insulin. It has been found that many tissues in the body secrete RLN, some of which enter the blood circulation as a systemic hormone, and some do not enter the circulation but only act as a local hormone to play an autocrine or paracrine role. RLN is beneficial to pregnancy maintenance and delivery. Dogs begin to secrete this hormone 10-15 days after pregnancy. At present, the domestic detection of dog pregnancy mainly relies on B-ultrasound, and the detection time of B-ultrasound is about 30 days.

[0003] The relaxin assay kit widely used in North America is used to test early pregnancy in dogs, but the commonly used RLN serological test uses enzyme-linked immunosorbent assay (ELISA) and fluorescent immunoassay. ELISA often uses porous polystyrene reaction plates as solid phase carriers, places the antigen or antibody to be tested on the surface of the solid phase carrier, and then uses enzyme-labeled antigen or antibody to react specifically with it. After adding the substrate, the result is judged by color reaction using an enzyme marker. This method is suitable for the detection of large quantities of samples, but its sensitivity and specificity are not very high, the degree of automation is low, and it is easy to make mistakes in operation and cause misjudgment of results. Fluorescent immunoassay is to mark fluorescent substances on antigens or antibodies and detect antigen-antibody reactions through fluorescent signals. This method has poor anti-interference, low sensitivity, and poor repeatability, resulting in inaccurate test results. Summary of the invention

[0004] The present invention aims to solve the problems of low sensitivity, poor specificity and poor repeatability of the existing enzyme-linked immunosorbent assay and fluorescent immunoassay in detecting early pregnancy in dogs, and provides a canine early pregnancy RLN detection kit and a detection method thereof with high test sensitivity, strong specificity and good repeatability.

[0005] The present invention adopts the following technical solution:

[0006] A canine early pregnancy RLN detection kit comprises a magnetic bead working solution, an enzyme working solution, a cleaning solution, a chemiluminescent substrate, a quality control product and a calibrator. The magnetic bead working solution contains tosyl magnetic beads labeled with canine RLN monoclonal antibodies, and the enzyme working solution contains canine RLN monoclonal antibodies labeled with alkaline phosphatase and an antibody blocker.

[0007] Furthermore, the mass concentration of the tosyl magnetic beads labeled with canine RLN monoclonal antibody in the magnetic bead working solution is 0.1-1 mg / ml, and the particle size of the tosyl magnetic beads is 1-4 μm.

[0008] Furthermore, the mass percentage concentration of canine RLN antibody labeled with alkaline phosphatase in the enzyme working solution is 0.05-0.4%, and the labeled mass ratio of canine RLN monoclonal antibody to alkaline phosphatase is 1:1-10:1.

[0009] Furthermore, the chemiluminescent substrate is (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane, and the cleaning solution is Tris buffer.

[0010] Furthermore, the antibody blocker is one of a canine heterophilic antibody blocker, an IgG blocker, an IgM blocker or a SAb blocker.

[0011] Furthermore, the mass percentage concentration of the blocker in the enzyme working solution is 0.1% to 1%.

[0012] Furthermore, the enzyme working solution also includes: 0.1-0.5 mol / L phosphate buffer, 0.15% by mass concentration of surfactant and 1% by mass concentration of protein, wherein the surfactant is Triton X-100, sodium dodecyl sulfate and Tween-20 mixed in equal proportions by mass.

[0013] Furthermore, the method for preparing the alkaline phosphatase-labeled canine RLN monoclonal antibody comprises the following steps:

[0014] Step 1: Activation of alkaline phosphatase: Add dimethylformamide (DMF) to the EMCS buffer, then add the EMCS buffer to the ALP preparation buffer, add the above mixture while stirring the ALP solution, and place in a water bath at 37.0±1℃ for 60min; desalt after the water bath, and recover the desalted solution;

[0015] Step 2: Reduction of alkaline phosphatase label:

[0016] (1) using a centrifugal desalting column to replace a canine RLN monoclonal antibody buffer, and recovering a solution after the buffer replacement;

[0017] (2) Prepare 0.3 M methylethanolamine MEA buffer, mix MEA buffer and canine RLN monoclonal antibody at a volume ratio of 1:9, and then incubate in a water bath at 37°C±1°C for 90 min. After the reaction is completed, centrifuge and desalt, recover the desalted solution, add activated alkaline phosphatase to the above solution, seal it, and let it stand at 2-8°C for 24 h. After the reaction is completed, add 0.1 M MEA solution to the above solution, mix well, and react at 37°C±1°C for 10 min. The solution is stored at 2-8°C.

[0018] Furthermore, the preparation method of the magnetic bead working solution comprises the following steps:

[0019] S1: Coating: Take tosyl magnetic beads, add boric acid solution, ammonium sulfate solution, dialyzed canine RLN monoclonal antibody and ProClin300 in sequence, mix well and seal with sealing film, place on a rotary shaker, react at 37℃±1℃ for 16-18 hours, canine RLN monoclonal antibody: tosyl magnetic beads are labeled at 20μg:1mg;

[0020] S2: Blocking and storage: Wash the magnetic beads, add BC solution and block at 36-38°C for 2-3 hours, prepare the magnetic bead marker to 25 mg / mL, and store at 2-8°C.

[0021] A detection method of a canine early pregnancy RLN detection kit, the steps in the determination process are as follows:

[0022] S1: Incubate the canine serum sample, enzyme working solution and magnetic bead working solution at a volume ratio of 2:5:5 at 36-38°C;

[0023] S2: subjecting the reaction solution obtained in S1 to magnetic separation and collecting magnetic beads;

[0024] S3: Rinse the magnetic beads with a cleaning solution to wash away unbound substances, add a chemiluminescent substrate, mix well, and allow for a full reaction;

[0025] S4: After the reaction is completed, the luminescence value is detected by a fully automatic chemiluminescence analyzer to obtain the concentration of canine RLN in the sample to be tested.

[0026] The advantages of the present invention are specifically as follows:

[0027] (1) In the detection process of the kit prepared by the present invention, canine RLN in the sample to be tested forms an antibody-antigen-antibody sandwich complex with the RLN monoclonal antibody labeled with the enzyme working solution and the RLN monoclonal antibody coated with magnetic beads. In this method, magnetic beads and RLN monoclonal antibodies labeled with alkaline phosphatase are added at the same time. The RLN antibodies on the magnetic beads and the RLN antibodies on the enzyme markers simultaneously and rapidly bind to different sites on the RLN antigen, so that the RLN antigen and the RLN monoclonal antibody are fully combined, thereby improving the detection sensitivity.

[0028] (2) The antibody blocker added to the enzyme working solution blocks the interference of heterophilic antibodies in the sample, shortening the window period of RLN detection, improving specificity, reducing false positives, and improving sensitivity.

[0029] (3) In the kit prepared by the present invention, the enzyme working solution reagent also includes surfactants: Triton X-100, sodium dodecyl sulfate and Tween-20. The surfactant can reduce the nonspecific effect of the antibody on the antigen, and is used to elute the unbound antibody, reduce nonspecific binding, improve specific recognition ability, and facilitate the binding of RLN antigen and antibody, thereby improving the sensitivity of the kit.

[0030] (4) The detection kit prepared by the present invention uses tosyl magnetic particles as solid phase carriers, which expands the reaction area and is easy to clean and remove non-specific binding, thereby improving the detection accuracy.

[0031] (5) The kit prepared by the present invention realizes automated monitoring by being used in conjunction with a fully automatic chemiluminescence analyzer, thereby avoiding errors caused by human operation and improving the detection speed, detection efficiency and detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a reaction principle diagram of the kit of the present invention for detecting canine RLN. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0034] The tosyl magnetic beads in the present invention were purchased from Thermo Fisher (Cat. No. 30110D).

[0035] Example 1: Preparation of canine RLN monoclonal antibody labeled with alkaline phosphatase,

[0036] The specific method includes the following steps:

[0037] S1: Activation of alkaline phosphatase:

[0038] (1) Activation of alkaline phosphatase: DMF was added to EMCS to prepare 80 mM EMCS buffer, and then the EMCS buffer was added to the ALP preparation buffer. The ALP solution was stirred while adding the above mixture, and the mixture was incubated in a water bath at 37.0±1°C for 60 minutes. After the water bath, desalination was performed and the desalted solution was recovered.

[0039] S2: ALP-mal marker restoration:

[0040] Alkaline phosphatase label reduction: use a centrifugal desalting column to replace the canine RLN monoclonal antibody buffer, and recover the solution after the buffer replacement; prepare 0.3M MEA buffer: mix the MEA buffer and canine RLN monoclonal antibody in a volume of 1:9 and place in a water bath at 37℃±1℃ for 90min. After the reaction, centrifuge and desalt, recover the desalted solution, add the activated alkaline phosphatase to the above solution, seal it, and let it stand at 2-8℃ for 24h. After the reaction is completed, add 0.1M MEA solution to the above solution, mix well and react at 37℃±1℃ for 10 minutes. The solution is stored at 2-8℃.

[0041] Embodiment 2:

[0042] Preparation method of canine RLN monoclonal antibody coated with tosyl magnetic beads,

[0043] The specific steps include:

[0044] (1) Coating: Canine RLN monoclonal antibody: tosyl magnetic beads are labeled at a mass ratio of 20 μg:1 mg. Take tosyl magnetic beads, add BA (filled to 5X) μL, BB (1 / 3*5X) μL, dialyzed canine RLN monoclonal antibody X μL, BD 1 / 100*5*X μL in sequence, mix well, seal with sealing film, place on a rotary shaker, and react at 36-38°C for 16-18 hours;

[0045] (2) Blocking: After the reaction is completed, wash the magnetic beads once with BC solution, then add BC solution and block at 37±1℃ for 2-3 hours.

[0046] (3) Storage: Add BC solution to prepare the magnetic bead marker to 25 mg / mL and store at 2-8°C.

[0047] Embodiment three:

[0048] A canine early pregnancy test kit, comprising a magnetic bead working solution, an enzyme working solution, a cleaning solution, a chemiluminescent substrate, a calibration product and a quality control product;

[0049] The magnetic bead working solution contains tosyl magnetic beads labeled with canine RLN monoclonal antibody, the concentration of the tosyl magnetic beads is 0.1-1 mg / ml, the particle size of the magnetic particles is 1-4 μm, and the preparation method of the tosyl magnetic beads labeled with canine RLN monoclonal antibody is as shown in Example 2;

[0050] The enzyme working solution contains canine RLN monoclonal antibody labeled with alkaline phosphatase, the concentration of the canine RLN monoclonal antibody labeled with alkaline phosphatase is 0.05-0.4%, and the labeling ratio of canine RLN monoclonal antibody to alkaline phosphatase is 1:1-10:1; the preparation method of canine RLN monoclonal antibody labeled with alkaline phosphatase is as shown in Example 1;

[0051] The calibrators include calibrators 1 and calibrators 2 prepared by diluting canine RLN antigen with canine hormone-free serum; the quality control includes a quality control prepared by diluting canine RLN antigen with canine hormone-free serum;

[0052] The cleaning solution is Tris buffer; the chemiluminescent substrate is (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane.

[0053] Example 4: Kit for detecting early pregnancy in dogs, the principle is as follows Figure 1 As shown, the specific steps include:

[0054] S1: Add 20uL of the sample to be tested, mix with 50uL of enzyme working solution and 50uL of magnetic bead working solution, and incubate at 37±1℃. PLN in the sample to be tested forms an antibody-antigen-antibody sandwich complex with the labeled RLN monoclonal antibody in the enzyme working solution and the RLN monoclonal antibody coated with magnetic beads;

[0055] S2: subjecting the reaction solution obtained in S1 to magnetic separation and collecting magnetic beads;

[0056] S3: Rinse the magnetic beads with a cleaning solution to wash away unbound substances, add the chemiluminescent substrate, mix well, and allow for a full reaction;

[0057] S4: The luminescence value is determined by a fully automatic chemiluminescence analyzer to obtain the concentration of canine RLN in the sample to be tested. The added chemiluminescent substrate is (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane, AMPPD. The luminescence signal generated under the catalysis of the enzyme is received by the photon reading system, and the photomultiplier tube measures the number of photons generated by the reaction. The number of photons generated is proportional to the content of RLN in the sample to be tested. The concentration value of canine RLN in the sample to be tested is obtained by measurement.

[0058] Example 5: Adding a blocker and not adding a blocker

[0059] Based on the mother solution of the kit in Example 3, four blocking agents, namely, canine heterophilic antibody blocker, IgG blocker, IgM blocker and SAb blocker, were added to the enzyme working solution to prepare a kit, and the detection method of Example 4 was used to detect the RLN content in the sample to be tested. The results are shown in Table 1.

[0060] Samples to be tested: Canine RLN detection limit references (L1-L5) and false positive samples (H1-H4);

[0061] The concentration of the working solution of each component: the concentration of the magnetic bead working solution is 0.2 mg / ml, the concentration of the alkaline phosphatase-labeled canine RLN monoclonal antibody is 1:1000, and the incubation temperature is 37±1°C;

[0062] The control group was a test kit prepared in Example 3 of the present invention without adding a blocking agent.

[0063] Table 1 Performance results

[0064]

[0065]

[0066] Note: Reference range for early pregnancy in dogs: <3ng / mL for non-pregnant, ≥3ng / mL for pregnant;

[0067] As shown in Table 1, based on the kit of Example 3 (control experiment), after adding a blocker to the enzyme working solution, the detection sensitivity was significantly improved, and the addition of canine heterophilic antibody blocker had the best effect, which solved the problem of false positive.

[0068] Example 6: Adding a surfactant and not adding a surfactant

[0069] Based on the mother solution of the kit of Example 3, surfactants (Triton X-100, sodium dodecyl sulfate and Tween-20) were added to the enzyme working solution as sample 1, and the control group was the kit obtained in Example 3. The detection method of Example 4 was used, and other conditions remained unchanged to detect RLN in the sample to be tested. The results are shown in Table 2.

[0070] Table 2 Detection limit results

[0071]

[0072] Note: Reference range for early pregnancy in dogs: <3ng / mL when not pregnant, ≥3ng / mL when pregnant.

[0073] As can be seen from Table 2, after adding the surfactant, the detection sensitivity in the sample to be tested is significantly improved. The surfactant can reduce the nonspecific effect of the antibody on the antigen, and is used to elute the unbound antibody, reduce nonspecific binding, improve the specific recognition ability, and is conducive to the binding of the RLN antigen and the antibody, thereby improving the sensitivity of the kit.

[0074] Embodiment 7

[0075] On the basis of Example 3 of the present invention, a kit containing a canine heterophilic antibody blocker and a surfactant was prepared. The working solution concentrations of each component were as follows: the working solution concentration of magnetic beads was 0.2 mg / ml, the canine RLN monoclonal antibody labeled with alkaline phosphatase was 1:1000, the incubation temperature was 37±1°C, the blocker was a canine heterophilic antibody blocker, the surfactants were Triton X-100, sodium dodecyl sulfate and Tween-20, and the protein was bovine serum albumin.

[0076] (1) Evaluation of detection limit, precision, accuracy and batch variation.

[0077] The detection limit, precision, accuracy and batch difference of the kit prepared by the preparation method of the present invention were evaluated, and the results are shown in Table 3.

[0078] Sample to be tested: Canine RLN positive serum

[0079] Detection limit: Five samples with concentrations close to the detection limit were tested.

[0080] Precision: Three batches of canine early pregnancy test kits (chemiluminescence method) produced continuously by our company were used to test the samples to be tested, and the test was repeated 10 times. According to the formula CV = standard deviation (SD) / average value of 10 test values Calculate the coefficient of variation.

[0081] Accuracy: Use accuracy reference products with traceability as test samples. Each reference product is measured 3 times and the result is recorded as M. The deviation of the measurement result is calculated according to the formula: measurement deviation = (M-theoretical value) / theoretical value × 100%.

[0082] Batch difference: Use three batches of test kits to test the samples separately, repeat each batch 10 times, and calculate the average of the 30 test results And standard deviation (SD), according to the formula CV = standard deviation (SD) / average of 10 test values Calculate the coefficient of variation.

[0083] Table 3 Performance evaluation results

[0084]

[0085]

[0086] As shown in Table 3, the detection limits of the three batches of reagents are 0.03ng / ml, 0.04ng / ml, and 0.04ng / ml; the precision CV test results are 3.52%, 4.01%, and 3.48%, respectively, all less than 10%; the accuracy reference deviations are 4.51%, 3.89%, and 3.24%, respectively. The inter-batch difference test results of the three batches of reagents are 5.96%, less than 10%; from this data, it can be seen that the inter-batch difference of this kit is good and can meet the designed CV% requirement of less than 10%.

[0087] (2) Stability determination

[0088] The test kits of the same batch were placed at 2-8°C and 37±1°C for 7 days, then taken out and placed at room temperature for equilibrium. Under the same conditions, the samples to be tested were tested according to the requirements of canine early pregnancy test, and the data were recorded. The results are shown in Table 4.

[0089] Sample to be tested: Canine RLN positive serum

[0090] Table 4 Stability evaluation results

[0091]

[0092] As shown in the results of Table 4, the detection limit of the kit prepared by the present invention is 0.03 ng / mL after 7 days at 2-8°C and 37±1°C; the precision CV test results are 3.25% and 4.38% respectively; the deviations of the accuracy reference products are 3.86% and 4.89% respectively; all meet the requirements of the canine early pregnancy kit, and the results show that the kit prepared by the present invention has good stability.

Claims

1. A canine early pregnancy RLN detection kit, characterized in that: The components include magnetic bead working solution, enzyme working solution, cleaning solution, chemiluminescent substrate, quality control product and calibration product. The magnetic bead working solution contains tosyl magnetic beads labeled with canine RLN monoclonal antibody, and the enzyme working solution contains canine RLN monoclonal antibody labeled with alkaline phosphatase and antibody blocker.

2. The canine early pregnancy RLN detection kit according to claim 1, characterized in that: The mass concentration of the tosyl magnetic beads labeled with the canine RLN monoclonal antibody in the magnetic bead working solution is 0.1-1 mg / ml, and the particle size of the tosyl magnetic beads is 1-4 μm.

3. The canine early pregnancy RLN detection kit according to claim 1, characterized in that: The mass percentage concentration of the canine RLN antibody labeled with alkaline phosphatase in the enzyme working solution is 0.05-0.4%, and the labeling mass ratio of the canine RLN monoclonal antibody to the alkaline phosphatase is 1:1-10:

1.

4. The canine early pregnancy RLN detection kit according to any one of claims 1 to 3, characterized in that: The chemiluminescent substrate is (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane, and the cleaning solution is Tris buffer.

5. The canine early pregnancy RLN detection kit according to any one of claims 1 to 3, characterized in that: The antibody blocker is one of a canine heterophilic antibody blocker, an IgG blocker, an IgM blocker or a SAb blocker.

6. The canine early pregnancy RLN detection kit according to claim 5, characterized in that: The mass percentage concentration of the blocking agent in the enzyme working solution is 0.1% to 1%.

7. The canine early pregnancy RLN detection kit according to any one of claims 1 to 3, characterized in that: The enzyme working solution also includes: 0.1-0.5 mol / L phosphate buffer, 0.15% surfactant by mass and 1% protein by mass, wherein the surfactant is Triton X-100, sodium dodecyl sulfate and Tween-20 mixed in equal proportions by mass.

8. The canine early pregnancy RLN detection kit according to claim 3, characterized in that: The preparation method of the alkaline phosphatase-labeled canine RLN monoclonal antibody comprises the following steps: Step 1: Activation of alkaline phosphatase: Add dimethylformamide (DMF) to the EMCS buffer, then add the EMCS buffer to the ALP preparation buffer, add the above mixture while stirring the ALP solution, and place in a water bath at 37±1°C for 60 minutes; desalt after the water bath, and recover the desalted solution; Step 2: Reduction of alkaline phosphatase label: (1) using a centrifugal desalting column to replace a canine RLN monoclonal antibody buffer, and recovering a solution after the buffer replacement; (2) Prepare 0.3 M methylethanolamine MEA buffer, mix MEA buffer and canine RLN monoclonal antibody at a volume ratio of 1:9, and then incubate in a water bath at 37°C±1°C for 90 min. After the reaction is completed, centrifuge and desalt, recover the desalted solution, add activated alkaline phosphatase to the above solution, seal it, and let it stand at 2-8°C for 24 h. After the reaction is completed, add 0.1 M MEA solution to the above solution, mix well, and react at 37°C±1°C for 10 min. The solution is stored at 2-8°C.

9. The canine early pregnancy RLN detection kit according to claim 2, characterized in that: The preparation method of the magnetic bead working solution comprises the following steps: S1: Coating: Take tosyl magnetic beads, add boric acid solution, ammonium sulfate solution, dialyzed canine RLN monoclonal antibody and ProClin300 in sequence, mix well and seal with sealing film, place on a rotary shaker, react at 36-38°C for 16-18 hours, and label canine RLN monoclonal antibody: tosyl magnetic beads at 20μg:1mg; S2: Blocking and storage: Wash the magnetic beads, add BC solution and block at 37℃±1℃ for 2-3 hours, prepare the magnetic bead marker to 25mg / mL, and store at 2-8℃.

10. A detection method of a canine early pregnancy RLN detection kit, characterized in that: The steps in the determination process are as follows: S1: Incubate the canine serum sample, enzyme working solution and magnetic bead working solution at a volume ratio of 2:5:5 at 36-38°C; S2: subjecting the reaction solution obtained in S1 to magnetic separation and collecting magnetic beads; S3: Rinse the magnetic beads with a cleaning solution to wash away unbound substances, add a chemiluminescent substrate, mix well, and allow for a full reaction; S4: After the reaction is completed, the luminescence value is detected by a fully automatic chemiluminescence analyzer to obtain the concentration of canine RLN in the sample to be tested.

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