Alkaline phosphatase labeled antibody reagent

Abstract

The present invention relates to the field of biochemical detection technology, and in particular to an alkaline phosphatase labeled antibody reagent. The present invention provides an alkaline phosphatase labeled antibody reagent, in which the preservation solution can improve the stability of the components, especially improve the stability of the alkaline phosphatase labeled antibody, so that the reagent is used for the detection of the sample to be tested with better stability, sensitivity, accuracy, specificity and precision. In addition, in the present invention, the preparation of the alkaline phosphatase labeled antibody does not require a blocking agent in the activation stage, and the alkaline phosphatase is activated with 2-IT, and the antibody is activated with Sulfo-SMCC, thereby avoiding the interference of the activator with the ALP conformation, and the obtained enzyme-labeled antibody has good stability.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical detection, in particular to an alkaline phosphatase labeled antibody reagent. Background Art

[0002] Alkaline phosphatase (ALP or AKP) is one of the most commonly used marker enzymes in immunodiagnostic reagents. After the antibody is labeled with alkaline phosphatase (ALP), an immune reaction forms a solid-phase coated antibody-antigen to be tested-enzyme-labeled antibody complex. The alkaline phosphatase in the complex removes the phosphate group of the luminescent agent and emits light.

[0003] Alkaline phosphatase labeled antibody reagent is a reagent containing alkaline phosphatase labeled antibody and buffer, which has an important influence on the results of immunoassay. Studies have shown that the reasons why the reagent affects the test results are mainly related to the preparation method of alkaline phosphatase labeled antibody and the components of buffer. On the one hand, the commonly used methods for labeling alkaline phosphatase antibodies include EDC-NHS and SMCC labeling methods. The EDC-NHS labeling method has a long labeling time and poor stability of the conjugate. In the SMCC labeling method, 2-MEA and TCEP are commonly used to activate antibodies, and Sulfo-SMCC activates alkaline phosphatase. 2-MEA and TCEP activate antibodies, destroy the antibody structure, have a certain impact on stability, and the activation environment is relatively strict and the activation time is long. On the other hand, the preservation solution has a significant effect on the stability of alkaline phosphatase enzyme labeled antibodies. However, most of the commonly used preservation solutions contain similar protective agents such as glucose, glycerol, and polyethylene glycol polymers. There are many types of added, the reagent components are complex, and the protection effect is not obvious; and after adding these protective agents, the performance of the reagent may be affected.

[0004] At present, further research is needed on how to further improve the stability of alkaline phosphatase-labeled antibody reagents and thus improve the detection effect. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an alkaline phosphatase-labeled antibody reagent with good stability.

[0006] The invention provides an alkaline phosphatase labeled antibody reagent, which comprises an alkaline phosphatase labeled antibody mother solution and a preservation solution.

[0007] In the present invention, the preservation solution is composed of water and 10-100 mmol / L buffer salt, 2-20 g / L protein, 0.5-5 g / L surfactant, 0.1-0.5 mol / L salt, 0.5-5 g / L preservative, and 0.5-20 g / L protective agent; wherein the protective agent is glycine and sulfate.

[0008] The protective agent and the enzyme-labeled antibody mother solution of the present invention can be mixed or exist independently of each other, and the present invention does not limit this. The preservation solution of the present invention can exist at the same concentration as the working concentration, or at a concentration of 2 to 100 times the working concentration. The preservation solution of the reagent of the present invention does not contain glycine and sulfate, which work together with other components to significantly improve the stability of the ALP-labeled antibody. Wherein, the sulfate is sodium sulfate or potassium sulfate.

[0009] The invention uses common sodium sulfate and glycine as protective agents to effectively improve the stability of the alkaline phosphatase-labeled cardiac troponin I antibody during storage and use. The invention has simple components, is easy to prepare, and has low cost.

[0010] In the ALP labeled antibody reagent of the present invention, the concentration of the alkaline phosphatase labeled antibody prepared by the preparation method of the present invention is 0.5-5 ng / mL. In some embodiments of the present invention, the reagent of the alkaline phosphatase labeled antibody provided by the present invention is composed of water and 0.5-5 ng / mL alkaline phosphatase labeled antibody, 10-100 mmol / L buffer salt, 2-20 g / L protein, 0.5-5 g / L surfactant, 0.1-0.5 mol / L salt, 0.5-5 g / L preservative, and 0.5-20 g / L protective agent; the protective agent is glycine and sulfate. Experiments show that under the selection of the above reagents, the effect of the obtained preservation solution is better than the selection or concentration of other components. Wherein, the sulfate is sodium sulfate or potassium sulfate.

[0011] In the reagent of the present invention, the buffer salt is selected from any one of Tris, MES, MOPS, and HEPES; in the embodiment of the present invention, the buffer salt is Tris, and the concentration of the buffer salt is 50 mM.

[0012] In the reagent of the present invention, the protein is selected from any one of bovine serum albumin, casein, and gelatin, or a combination of two or more thereof. In an embodiment of the present invention, the protein is bovine serum albumin. The concentration of the protein is 10 g / L.

[0013] In the reagent of the present invention, the surfactant is selected from any one of Tween 20, Tween 40, Tween 80, Triton X-100, Triton X-405, and Brij-35, or a combination of two or more thereof. In an embodiment of the present invention, the surfactant is Brij-35. The concentration of the surfactant is 1 g / L.

[0014] In the reagent of the present invention, the salt is selected from any one or a combination of two or more of sodium chloride, magnesium chloride, zinc chloride and potassium chloride. In an embodiment of the present invention, the salt is a combination of sodium chloride, magnesium chloride and zinc chloride, wherein the sodium chloride concentration is 0.1 to 0.3 mol / L, and the preferred sodium chloride concentration is 0.15 mol / L. The magnesium chloride concentration is 1 to 10 mmol / L, and the preferred magnesium chloride concentration is 2.5 mmol / L. The zinc chloride concentration is 0.2 to 5 mmol / L, and the preferred zinc chloride concentration is 0.5 mmol / L.

[0015] In the reagent of the present invention, the preservative is selected from any one or a combination of two or more of sodium azide, PC300, chloramphenicol, and gentamicin; in the embodiment of the present invention, the preservative is sodium azide. The concentration of sodium azide is 1 g / L.

[0016] In the reagent of the present invention, the glycine concentration is 0.5-10 g / L, and the sodium sulfate concentration is 5-20 g / L. In an embodiment of the present invention, the glycine concentration is 0.5, 5 or 10 g / L, and the sulfate concentration is 5, 10 or 20 g / L. In the preservation solution of the present invention, the sulfate is sodium sulfate or potassium sulfate.

[0017] The pH value of the reagent of the present invention is 7.0-8.0, and the preferred pH value is 7.40.

[0018] In the reagent of the present invention, the preparation method of the alkaline phosphatase labeled antibody comprises:

[0019] Activating alkaline phosphatase with 2-IT to obtain activated alkaline phosphatase;

[0020] Activating the antibody with Sulfo-SMCC to obtain activated antibody;

[0021] The activated alkaline phosphatase and the activated antibody are mixed, N-ethylmaleimide is added, and then the alkaline phosphatase-labeled antibody is obtained by blocking.

[0022] The present invention uses 2-IT to activate alkaline phosphatase, so that it carries a thiol group connected to the antibody after Sulfo-SMCC activation, so as to achieve the purpose of coupling the antibody and alkaline phosphatase. The method does not need to add a blocking solution in either the antibody activation stage or the alkaline phosphatase activation stage, thus reducing the operation steps and saving time. Compared with the scheme of using Sulfo-SMCC to activate ALP, the alkaline phosphatase labeled antibody prepared by the present invention is more stable and is conducive to improving the detection effect.

[0023] The method described in the present invention does not limit the source of alkaline phosphatase or antibody, and experiments show that the source of the raw materials has no effect on the effect.

[0024] In some embodiments, the alkaline phosphatase is treated with a desalting column before activation.

[0025] In the activation step of alkaline phosphatase, the buffer is an enzyme labeling solution 1, which includes water and 1g / kg to 5g / kg NaH2PO4·2H2O, 25g / kg to 30g / kg Na2HPO4·12H2O, 8g / kg to 10g / kg NaCl, and 0.9g / kg to 1g / kg Na2EDTA·2H2O. In some embodiments, the enzyme labeling solution 1 is composed of water and 3g / kg NaH2PO4·2H2O, 29g / kg Na2HPO4·12H2O, 9g / kg NaCl, and 0.93g / kg Na2EDTA·2H2O. The pH value of the enzyme labeling solution 1 is 8.0.

[0026] In the activation step of alkaline phosphatase, the mass ratio of alkaline phosphatase to 2-IT is 100:(1-5). In some embodiments, the mass ratio of alkaline phosphatase to 2-IT is 100:2.

[0027] Specifically, the activation of alkaline phosphatase includes dissolving alkaline phosphatase with enzyme labeled solution 1 to a concentration of 5 mg / mL to obtain an ALP solution, and dissolving 2-IT with enzyme labeled solution 1 to a concentration of 2 mg / mL to obtain a 2-IT solution. The ALP solution and the 2-IT solution are mixed and allowed to stand at 20 to 25° C. for 0.1 to 1 h. In some embodiments, the activation condition is standing at 22° C. for 30 min. The activated alkaline phosphatase is purified by a desalting column to obtain an activated ALP solution.

[0028] The method of the present invention can be applied to the labeling of various antibodies. In the present invention, the antibody can be selected from any one of cardiac troponin I antibody, interleukin-6 antibody or myoglobin antibody. In the embodiment of the present invention, cardiac troponin I antibody is taken as an example to verify the effect of the preparation method.

[0029] In some embodiments, the antibody is treated with a desalting column before activation.

[0030] In the activation step of the antibody, the buffer is an enzyme labeling solution 2, which includes water and 1g / kg to 5g / kg NaH2PO4·2H2O, 25g / kg to 30g / kg Na2HPO4·12H2O, 15g / kg to 20g / kg NaCl, and 1g / kg to 5g / kg Na2EDTA·2H2O. In some embodiments, the enzyme labeling solution 2 is composed of water and 3g / kg NaH2PO4·2H2O, 29g / kg Na2HPO4·12H2O, 18g / kg NaCl, and 3.72g / kg Na2EDTA·2H2O. The pH value of the enzyme labeling solution 1 is 8.0.

[0031] In the antibody activation step, the mass ratio of the antibody to Sulfo-SMCC is 100:(5-10). In some embodiments, the mass ratio of the antibody to Sulfo-SMCC is 100:8.5.

[0032] Specifically, the activation of the antibody includes dissolving the antibody in enzyme labeled solution 2 to a concentration of 4 mg / mL to obtain an antibody solution, and dissolving Sulfo-SMCC in enzyme labeled solution 2 to a concentration of 5 mg / mL to obtain a Sulfo-SMCC solution. The antibody solution and the Sulfo-SMCC solution are mixed and allowed to stand at 20 to 25° C. for 0.1 to 1 h. In some embodiments, the activation condition is to stand at 22° C. for 30 min. The activated antibody is purified by a desalting column to obtain an activated antibody solution.

[0033] In the present invention, the activated ALP solution and the activated antibody solution are mixed. In this step, the mass ratio of activated ALP to activated antibody is (0.5-2):1.

[0034] After the activated ALP solution and the activated antibody solution are mixed, they are allowed to stand at 20-25° C. for 0.1-3 hours. In a specific embodiment, after the activated ALP solution and the activated antibody solution are mixed, they are allowed to stand at 22° C. for 60 hours.

[0035] In the present invention, the activated ALP solution and the activated antibody solution are mixed and left to stand at 22° C. for 60 hours before adding N-ethylmaleimide (NEM) and then blocking. In the present invention, blocking solution is added during the blocking stage to effectively avoid nonspecific binding, thereby improving the sensitivity, precision, specificity, accuracy and stability of the reagent.

[0036] In the present invention, the mass ratio of NEM to ALP is (5-20): 100. The reaction condition after adding NEM is to stand at 20-25°C for 5-10 minutes. In some embodiments, the reaction condition after adding NEM is to stand at 22°C for 10 minutes.

[0037] In the present invention, the closed blocking agent is ethanolamine. The ethanolamine is an enzyme labeling solution 3. The enzyme labeling solution 3 includes water and 25g / kg to 26g / kg Na2HPO4·12H2O, 4g / kg to 5g / kg NaH2PO4·2H2O, and 5g / kg to 10g / kg NaCl. In some embodiments, the enzyme labeling solution 3 is composed of water and 25.78g / kg Na2HPO4·12H2O, 4.36g / kgNaH2PO4·2H2O, and 8.78g / kg NaCl. The pH value of the enzyme labeling solution 1 is 7.4. In some embodiments, the mass ratio of the ethanolamine to ALP is (1 to 2): 1. The conditions for the closed reaction are to stand at 20 to 25°C for 10 to 20 minutes. In some embodiments, the conditions for the closed reaction are to stand at 22°C for 10 minutes.

[0038] In the method for preparing the ALP labeled antibody of the present invention, the steps of ultrafiltration and preparing a mother solution are further included after the blocking.

[0039] In the present invention, the molecular weight of the ultrafiltration membrane is 30 kDa, and the concentration of the ALP-labeled antibody after ultrafiltration is 0.2 mg / mL. The concentration of the ALP-labeled antibody after ultrafiltration is adjusted with enzyme labeling solution 3.

[0040] In the present invention, the preparation of the mother solution includes adding glycerol to the ultrafiltered liquid, wherein the volume of the glycerol is equal to the volume of the ultrafiltered liquid.

[0041] The labeling process provided by the present invention has a short labeling time, simple operation, and improved activation efficiency. The reagent provided by the present invention uses the alkaline phosphatase labeled antibody as the detection antibody, has better stability, and can be used for detection to obtain higher sensitivity, accuracy, specificity and precision.

[0042] The present invention also provides an immunological detection reagent, which comprises the reagent of the present invention, a coated antibody reagent and a luminescent agent.

[0043] The present invention also provides an immunological detection method, which comprises detecting an antigen or antibody to be detected using the immunological detection reagent of the present invention.

[0044] The immunological detection method of the present invention includes detecting disease markers using immunological methods for the purpose of diagnosis, and may also include detecting other antigens to be detected for non-diagnostic purposes.

[0045] The present invention provides an alkaline phosphatase labeled antibody reagent, wherein the preservation solution in the reagent can improve the stability of the components, especially improve the stability of the alkaline phosphatase labeled antibody, so that the reagent has better stability, sensitivity, accuracy, specificity and precision for the detection of the antigen to be tested. In addition, in the present invention, the preparation of the alkaline phosphatase labeled antibody does not require a blocking agent in the activation stage, 2-IT is used to activate the alkaline phosphatase, and Sulfo-SMCC is used to activate the antibody, thereby avoiding the interference of the activator on the ALP conformation, and the obtained enzyme-labeled antibody has good stability. DETAILED DESCRIPTION

[0046] The present invention provides alkaline phosphatase labeled antibody reagents, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0048] Example 1 Preparation of Alkaline Phosphatase Labeled Cardiac Troponin I Antibody:

[0049] 1. Prepare reagents according to Tables 1 to 4:

[0050] Table 1 Enzyme labeling solution 1

[0051] Material Name unit Dosage <![CDATA[NaH2PO4·2H2O]]> g 3.00 <![CDATA[Na2HPO4·12H2O]]> g 29.00 NaCl g 9.00 Ethylenediaminetetraacetic acid disodium salt dihydrate g 0.93

[0052] Weigh each reagent into a clean beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust the pH to 8.0 with 5M NaOH solution, and add purified water to 1.0 kg. Filter with a 0.22 μm filter membrane and place at 2-8°C for later use.

[0053] Table 2 Enzyme labeling solution 2

[0054] Material Name unit Dosage <![CDATA[NaH2PO4·2H2O]]> g 3.00 <![CDATA[Na2HPO4·12H2O]]> g 29.00 NaCl g 18.00 Ethylenediaminetetraacetic acid disodium salt dihydrate g 3.72

[0055] Weigh each reagent into a clean beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust the pH to 8.0 with 5M NaOH solution, and add purified water to 1.0 kg. Filter with a 0.22 μm filter membrane, and place at 2-8 °C for later use.

[0056] Table 3 Enzyme labeling buffer 3

[0057] materials unit Dosage <![CDATA[Na2HPO4·12H2O]]> g 25.78 <![CDATA[NaH2PO4·2H2O]]> g 4.36 NaCl g 8.78

[0058] Weigh the above reagents into a clean beaker, add 0.8 kg of ultrapure water, stir thoroughly to dissolve completely, adjust pH to 7.4 with 5M NaOH or 5M HCl solution; add purified water to 1.0 kg, filter with a 0.22 μm filter head and set aside.

[0059] Table 4 Blocking solution

[0060] Material Name unit Dosage Ethanolamine g 2.50

[0061] Weigh each reagent into a glass bottle, add 250 mL of enzyme labeling solution 2, stir until completely dissolved, adjust the pH to 7.40 with 5 M NaOH solution, filter with a .22 μm filter membrane, and store at 2-8 °C for later use.

[0062] 2. Prepare alkaline phosphatase-labeled cardiac troponin I antibody according to the following steps:

[0063] 1) Take 100 μg of alkaline phosphatase, treat it with a desalting column, adjust the concentration to 5 mg / mL with enzyme labeling solution 1, add 2 μL of 2-IT solution (prepared with enzyme labeling solution 1, concentration is 2 mg / mL), vortex mix, let stand at 22°C for 30 min, and purify with a desalting column;

[0064] 2) Take 100 μg of antibody, treat with desalting column, adjust the concentration to 4 mg / mL with enzyme labeling solution 2, add 1.7 μL of Sulfo-SMCC solution (prepared with ultrapure water, concentration is 5 mg / mL), vortex to mix, stand at 22°C for 30 min, and purify with desalting column;

[0065] 3) Mix the activated alkaline phosphatase and antibody, let stand at 22°C for 60 min, add 5 μL of N-ethylmaleimide (prepared in enzyme label solution 2, concentration is 10 mg / mL), let stand at 22°C for 10 min, add 10 uL of blocking solution, let stand at 22°C for 10 min;

[0066] 4) After blocking, transfer to a 30K ultrafiltration tube, add enzyme labeling solution 3 to 400 μL, 9000 rpm / min, 25 min;

[0067] 5) After centrifugation, the enzyme-labeled antibody mother solution was aspirated, and the enzyme-labeled solution 3 was added to make up to 250 μL, and 250 μL of glycerol was added to obtain a 0.2 mg / mL enzyme-labeled antibody mother solution, which was stored at -20°C.

[0068] Embodiment 2:

[0069] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0070] Table 5 Preservative solution

[0071] Material Code unit Dosage(1kg) Tris g 6.06 NaCl g 9.00 BSA g 10.00 Brij-35 g 1.00 <![CDATA[NaN3]]> g 1.00 Glycine g 5.00 Sodium sulfate g 10.00 2M magnesium chloride solution mL 1.25 0.2M zinc chloride solution mL 2.50

[0072] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0073] Example 3

[0074] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0075] Table 6 Preservative solution

[0076] Material Code unit Dosage(1Kg) Tris g 6.06 NaCl g 9.00 BSA g 10.00 Brij-35 g 1.00 <![CDATA[NaN3]]> g 1.00 Glycine g 0.50 Sodium sulfate g 20.00 2M magnesium chloride solution mL 1.25 0.2M zinc chloride solution mL 2.50

[0077] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0078] Example 4

[0079] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0080] Table 7 Preservative solution

[0081] Material Code unit Dosage(1Kg) Tris g 6.06 NaCl g 9.00 BSA g 10.00 Brij-35 g 1.00 <![CDATA[NaN3]]> g 1.00 Glycine g 10.00 Sodium sulfate g 5.00 2M magnesium chloride solution mL 1.25 0.2M zinc chloride solution mL 2.50

[0082] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0083] Example 5

[0084] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0085] Table 8 Preservative solution

[0086]

[0087]

[0088] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0089] Comparative Example 1:

[0090] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0091] Table 9 Preservative solution

[0092] Material Code unit Dosage(1Kg) Tris g 6.06 NaCl g 9.00 BSA g 10.00 Brij-35 g 1.00 <![CDATA[NaN3]]> g 1.00 2M magnesium chloride solution mL 1.25 0.2M zinc chloride solution mL 2.50

[0093] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0094] Comparative Example 2:

[0095] The components and contents of the storage solution for alkaline phosphatase labeled conjugate are as follows:

[0096] Table 10 Preservative solution

[0097]

[0098]

[0099] Weigh each reagent in a beaker, add 0.8 kg of purified water, stir until completely dissolved, adjust pH to 7.40 with 5M NaOH solution, add purified water to 1.0 kg, filter with a 0.22 μm filter membrane, and store at 2-8°C for later use.

[0100] Experimental Example 1: Stability Verification:

[0101] The enzyme-labeled reagent was prepared by adding the alkaline phosphatase-labeled cardiac troponin I antibody stock solution prepared in Example 1 to the preservation solution described in Examples 2 to 5 and Comparative Examples 1 to 2.

[0102] Each of the above reagents was divided into two portions, one was placed at 37°C for accelerated aging for 7 days, and the other was placed at 2-8°C. After 7 days, the reagent placed at 2-8°C was used as a control, and the same sample was tested with two portions of enzyme-labeled reagents simultaneously. The test results are shown in Table 11:

[0103] Table 11 Stability test results

[0104]

[0105]

[0106] From the test results, it can be seen that the alkaline phosphatase conjugate prepared with the preservation solution of each embodiment is accelerated at 37°C for 7 days, and the luminescence value of the cTnI test decreases within 3%; and the protective agent content has little effect on stability. From the comparative results, it can be seen that without adding protective agents glycine and sodium sulfate (potassium sulfate), the luminescence value decreases by more than 15% at 37°C for 7 days; adding protective agents such as sucrose, polyethylene glycol 4000, and glycerol, the luminescence value decreases by more than 15% at 37°C for 7 days.

[0107] Experimental Example 2: Precision Verification:

[0108] High and low concentrations of cardiac troponin I repeatability reference substances (R1 and R2) were taken and tested 10 times respectively with the alkaline phosphatase labeled cardiac troponin I antibody reagent prepared in the preservation solution of Example 2, and the coefficient of variation (CV) was calculated.

[0109] Table 12 Precision verification results

[0110]

[0111] The results showed that the reagents prepared with this preservation solution had a small coefficient of variation between multiple tests and had good precision.

[0112] Experimental Example 3: Accuracy Verification:

[0113] The alkaline phosphatase-labeled cardiac troponin I antibody reagent prepared in the preservation solution of Example 2 was used to test the traceable corporate reference product, and the test deviation was calculated.

[0114] Table 13 Accuracy verification results

[0115]

[0116]

[0117] The results showed that the reagents prepared with this preservation solution had small deviations between multiple tests of the reference material and had good accuracy.

[0118] Experimental Example 3 Blank Limit Verification:

[0119] The zero concentration calibrator was tested 20 times with the alkaline phosphatase labeled cardiac troponin I antibody reagent prepared in the preservation solution of Example 2, and the average luminescence value was calculated. and standard deviation SD, Substituting the concentration-luminescence value between zero concentration and the adjacent calibrator into the equation, the resulting concentration value is the blank limit.

[0120]

[0121]

[0122] The results show that the blank limit of the reagent test of the present invention is 0.001 ng / mL, and the reagent has good sensitivity.

[0123] Experimental Example 4 Specificity Verification:

[0124] Cardiac troponin C (cTnC), cardiac troponin T (cTnT), and skeletal muscle troponin I (sTnI) at a concentration of 1000 ng / mL were added to the zero concentration calibrator, and the alkaline phosphatase-labeled cardiac troponin I antibody reagent prepared in the preservation solution of Example 2 was used to test the results.

[0125] Table 14 Specificity verification results

[0126] Cross Concentration (ng / mL) Calculated concentration (ng / mL) Cross-reaction rate cTnC 1000 0.005 0.0005％ TvT 1000 0.008 0.0008％ i 1000 0.006 0.0006％

[0127] The results show that the cross-reaction rates of the reagents of the present invention to the test results of analogs are all less than 0.1%, indicating that the three analogs have no interference with the reagents and can achieve accurate detection of cardiac troponin I.

[0128] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reagent for labeling an alkaline phosphatase antibody, characterized in that: It is composed of water and 0.5~5 ng / mL alkaline phosphatase-labeled antibody, 10~100mmol / L Tris, 2~20g / L bovine serum albumin, 0.5~5 g / L Brij-35, 0.1~0.3mol / L sodium chloride, 1~10mmol / L magnesium chloride, 0.2~5mmol / L zinc chloride, 0.5~5 g / L sodium azide, 0.5~10g / L glycine and 5~20g / L sulfate.

2. The reagent according to claim 1, characterized in that The sulfate is sodium sulfate or potassium sulfate.

3. The reagent according to claim 1, characterized in that The preparation method of the alkaline phosphatase labeled antibody comprises: Activating alkaline phosphatase with 2-IT to obtain activated alkaline phosphatase; Activating the antibody with Sulfo-SMCC to obtain activated antibody; The activated alkaline phosphatase and the activated antibody are mixed, N-ethylmaleimide is added, and then the alkaline phosphatase-labeled antibody is obtained by blocking.

4. The reagent according to claim 3, characterized in that The antibody includes any one of cardiac troponin I antibody, interleukin-6 antibody or myoglobin antibody.

5. The reagent according to claim 3, characterized in that The blocking agent is ethanolamine.

6. The reagent according to claim 3, characterized in that After the sealing, the steps of ultrafiltration and preparation of mother solution are also included; The preparation of the mother solution comprises adding glycerol to the liquid after ultrafiltration.

7. An immunological detection reagent, characterized in that: The method comprises the reagent according to any one of claims 1 to 6, a coated antibody reagent and a luminescent agent.

8. An immunological detection method, characterized in that: The method comprises detecting the antigen or antibody to be detected using the immunological detection reagent according to claim 7.

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