A labeling method for improving sensitivity and stability of a kit and application and kit thereof

Abstract

The application relates to the technical field of biological detection, and discloses a labeling method for improving sensitivity and stability of a kit and application and the kit, which comprises the following steps: reacting Traut's reagent with an antibody to obtain a thiolated antibody by hydroxylamine deprotection; incubating the thiolated antibody with a blocking solution containing mPEG-NHS; reacting a heterotypic bifunctional crosslinking agent with alkaline phosphatase to obtain activated alkaline phosphatase after quenching; coupling: incubating the blocked thiolated antibody with the activated alkaline phosphatase in a coupling buffer to obtain an enzyme-labeled antibody. The application realizes directional labeling of alkaline phosphatase polymer on the antibody, improves the affinity of the antibody-alkaline phosphatase complex, has short process time, simple steps, small risk points in the process amplification process, can be controlled, and can reduce cost and increase efficiency. The scheme does not need to use dangerous reagents, the method has strong universality, wide application range, and improved sensitivity and stability performance of the corresponding product kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a labeling method for improving sensitivity and stability of a kit and application and a kit. BACKGROUND

[0002] Under the background of rapid development of modern life science and medical detection technology, alkaline phosphatase (AP) labeled antibody kit in the field of immunoassay has become an indispensable tool in many fields such as clinical diagnosis, food safety detection and environmental monitoring due to its high specificity and sensitivity. As a classic labeled enzyme, alkaline phosphatase can specifically catalyze the color reaction or chemiluminescence reaction of the substrate, and through precise detection of the reaction signal intensity, high-precision quantitative and qualitative analysis of target antigens or antibodies can be realized.

[0003] Although alkaline phosphatase labeled antibody kit is widely used, there are still many technical bottlenecks to be overcome in the existing technology. In terms of sensitivity, the content of target biomarkers in clinical samples is often in trace amounts, and the concentration of tumor markers in the early stages of some diseases may be as low as pg / mL level, while the detection lower limit of traditional kits is usually in ng / mL level, which is difficult to meet the detection needs of ultra-micro substances. This lack of sensitivity can easily lead to the failure to effectively identify low-concentration target antigens or antibodies. According to relevant clinical statistics, the missed detection rate caused by insufficient sensitivity of the kit in early screening of tumors is as high as 15%-20%, which seriously affects the early diagnosis and intervention opportunity of the disease.

[0004] Encephalitis is a neurological dysfunction caused by diffuse or multiple inflammatory lesions in the brain parenchyma, and its pathological changes are mainly in the gray matter and neurons, and can also involve white matter and blood vessels. Autoimmune encephalitis (AE) refers to a group of encephalitides mediated by autoimmune mechanisms, and its main clinical symptoms include mental behavior abnormalities, cognitive impairment, recent memory loss, seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, autonomic dysfunction, etc. The diagnosis of AE first requires comprehensive analysis of the patient's clinical manifestations, cerebrospinal fluid examination, neuroimaging and electroencephalogram results to determine whether the patient has encephalitis, and then select AE-related antibody detection for diagnosis. Therefore, the stability of the encephalitis kit cannot be ignored.

[0005] In the field of clinical application of chemiluminescent immunoassay and in vitro diagnosis, alkaline phosphatase-labeled antibody reagent kit plays a core role. The activity of alkaline phosphatase is extremely sensitive to external environment, and temperature fluctuation, pH change and long-term storage will significantly affect its catalytic activity. This directly leads to the shortening of the shelf life of the kit, and the repeatability and consistency of the detection results are difficult to guarantee. In addition, the antibody labeling process also has technical defects. The traditional glutaraldehyde cross-linking labeling method can cause the activity loss of antibodies to be 30%-40%, and the shedding rate of the label during storage is as high as 25%, further weakening the detection efficiency of the kit. The glutaraldehyde cross-linking labeling method does not require complex pretreatment, and can directly react with the amino groups on the alkaline phosphatase and the antibody, which is simple to operate and easy to master. However, glutaraldehyde can randomly react with multiple amino groups of proteins, leading to random cross-linking within or between protein molecules, affecting the spatial structure and activity of the protein, and reducing the activity and detection sensitivity of the labeled product. At the same time, the Schiff base bond formed is unstable and is easily broken by environmental factors, and the label is easily shed during storage and transportation, shortening the shelf life of the kit and reducing the repeatability of the detection results. Moreover, due to the random cross-linking characteristics, it is difficult to accurately control the labeling site and the number of alkaline phosphatase and antibody, resulting in non-uniformity of the labeled product and affecting the detection accuracy.

[0006] In order to solve the above problems, domestic and foreign research institutions and enterprises actively carry out research and try to improve the performance of the kit by optimizing the labeling process, improving the buffer system and adding protective agents. For example, carbonic diamide method is used to replace glutaraldehyde method for labeling, which can reduce the activity loss rate of antibodies to about 20%, but still has the following problems: (1) low reaction efficiency: the cross-linking reaction rate of carbonic diamide is relatively slow, and side reactions such as self-polymerization may occur during the reaction, reducing the utilization rate of cross-linking agent and the labeling efficiency; (2) the stability of the amide bond formed by cross-linking is general, and may hydrolyze under certain conditions, affecting the long-term stability of the labeled product and requiring higher storage conditions for the kit; (3) although the reaction is relatively mild, the specificity of the labeling site is still not high enough, which may cause non-specific cross-linking and affect the uniformity of the labeled product and the detection results.

[0007] Adding protective agents such as bovine serum albumin (BSA) in the buffer system can delay the decline of enzyme activity to some extent, but has limited effect on improving sensitivity.

[0008] Therefore, the present application is proposed. SUMMARY

[0009] The prior art has problems of poor sensitivity and stability of alkaline phosphatase labeled antibody kit, in order to solve the above technical problems, the application provides a labeling method for improving the sensitivity and stability of the kit and application and the kit, SATP is used as an antibody thiolation reagent, which can protect the thiol group by acetylation to avoid its oxidation inactivation, then mPEG-NHS is used to specifically block the residual active amino site of the antibody, which does not interfere with the subsequent coupling of thiol and maleimide group, and the hydrophilicity of the PEG chain can reduce non-specific adsorption, improve hydrophilicity, inhibit thiol oxidation, and improve the stability of the labeled product, finally, the PEG flexible chain of NHS-PEG-maleimide and the PEG chain of mPEG-NHS cooperatively form a "double flexible support", which not only reduces the steric hindrance, but also improves the water solubility of the coupling product and the diffusion efficiency of the chemiluminescent substrate, further strengthens the enzyme catalytic luminescence intensity, and improves the sensitivity of detection.

[0010] The application is realized by the following technical solutions:

[0011] In a first aspect, the application provides a labeling method for improving the sensitivity and stability of the kit, comprising the following steps:

[0012] S1, antibody thiolation: the antibody is treated by using a desalting column, the storage solution is replaced by buffer 1, Traut's reagent is reacted with the antibody in the buffer, and the thiolated antibody with 2-3 free thiol groups per molecule is obtained by hydroxylamine deprotection;

[0013] S2, targeted blocking: the thiolated antibody of step S1 is incubated with a blocking solution containing mPEG-NHS at pH 7.0-7.2 for 20-30 minutes;

[0014] S3, alkaline phosphatase flexible activation: the alkaline phosphatase is treated by using a desalting column, the storage solution is replaced by buffer 2, the hetero-bifunctional crosslinking agent is reacted with the alkaline phosphatase in the buffer, and the activated alkaline phosphatase containing 3-4 active maleimide groups per molecule is obtained after quenching;

[0015] S4, coupling and purification: the blocked thiolated antibody is incubated with the activated alkaline phosphatase in a coupling buffer at 25 DEG C for 2 hours, and the enzyme-labeled antibody is obtained by gel filtration column purification.

[0016] In a certain specific embodiment, in step S1, the Traut's reagent is SATP, which is dissolved in buffer 1 and then connected with the amino group on the surface of the antibody, the reaction time is 0.5-1 h, and the reaction temperature is room temperature. After the reaction is completed, it is stored at 2-8 DEG C for standby use. The molar concentration ratio of SATP to antibody is 10-50:1, and the buffer 1 is a neutral phosphate buffer containing 1% ethylenediaminetetraacetic acid.

[0017] In a specific embodiment, in step S1, the final concentration of the hydroxylamine solution used for deprotection of hydroxylamine is 50 mM.

[0018] In a specific embodiment, in step S1, the antibody is a murine monoclonal antibody.

[0019] In a specific embodiment, in step S2, the blocking solution comprises PBS, EDTA, glycerol and mPEG-NHS. The specific composition can be as follows: 50 mM PBS, 1 mM EDTA, 5% glycerol and 5 mM mPEG-NHS.

[0020] In a specific embodiment, in step S3, the heterobifunctional crosslinking agent is an NHS-PEG-maleimide crosslinking agent.

[0021] In a specific embodiment, in step S3, the NHS-PEG-maleimide crosslinking agent is Maleimide-PEG2-NHS, Maleimide-PEG4-NHS or Maleimide-PEG8-NHS, preferably Maleimide-PEG4-NHS, abbreviated as SM(PEG)4.

[0022] In a specific embodiment, in step S3, the molar ratio of the heterobifunctional crosslinking agent to alkaline phosphatase is 50:1 to 80:1.

[0023] In a specific embodiment, in step S3, the buffer 2 is a Tris system buffer containing magnesium ions and zinc ions.

[0024] In a specific embodiment, in step S4, the molar ratio of the thiolated antibody to the activated alkaline phosphatase is 10:1 to 30:1, the reaction temperature is room temperature, and the reaction time is 1 hour.

[0025] The present application uses SATP as an antibody thiolation reagent, which can protect the thiol group by acetylation to avoid oxidation inactivation; then uses mPEG-NHS to specifically block the residual active amino site of the antibody, which does not interfere with the subsequent coupling of thiol and maleimide group, and the hydrophilicity of PEG chain can reduce non-specific adsorption, improve hydrophilicity, inhibit thiol oxidation, and improve the stability of the labeled product; finally, the PEG flexible chain of NHS-PEG-maleimide and the PEG chain of mPEG-NHS cooperatively form a "double flexible support", which not only reduces the steric hindrance, but also improves the water solubility of the conjugate and the diffusion efficiency of the chemiluminescent substrate, further strengthens the enzyme catalytic luminescence intensity, and improves the sensitivity of detection.

[0026] In a second aspect, the application provides application of the labeling method in chemiluminescence immunoassay.

[0027] In a third aspect, the application provides a chemiluminescence immunoassay kit with high sensitivity and stability, comprising the enzyme-labeled antibody prepared by the labeling method.

[0028] Compared with the prior art, the application has the following advantages and beneficial effects:

[0029] 1. The labeling method and application and kit for improving sensitivity and stability of a kit provided by the embodiment of the application utilize the combination design of "SATP (acetylated protected sulfhydryl) + mPEG-NHS (targeted blocked amino) + SM (PEG)4 (flexible activated AP)" reagents, which solves the problems of coupling efficiency and stability caused by the three defects of "sulfhydryl oxidation, steric hindrance and non-specific adsorption", and integrates the chemiluminescence detection to be used in an antibody-AP coupling kit, fully utilizes the high sensitivity characteristics of the chemiluminescence method, and amplifies the technical advantages, so that the detection lower limit can be as low as pg, which is significantly better than the conventional "colorimetric method + conventional reagent" system.

[0030] 2. The labeling method and application and kit for improving sensitivity and stability of a kit provided by the embodiment of the application adopt SATP activated antibodies with acetylated protected sulfhydryl, which can avoid sulfhydryl oxidation inactivation, thereby solving the problems of easy oxidation of sulfhydryl and low coupling efficiency of conventional sulfhydryl reagents.

[0031] 3. The labeling method and application and kit for improving sensitivity and stability of a kit provided by the embodiment of the application adopt mPEG-NHS blocking liquid specially designed for SATP activated antibodies + chemiluminescence detection, which can be matched with the structural characteristics of the antibodies after SATP deprotection, blocks only the residual amino groups on the surface of the antibodies without interfering with the sulfhydryl groups, and can avoid the coupling of the sulfhydryl groups with the maleimide groups in the subsequent process. Meanwhile, the PEG chain contained in SATP has good hydrophilicity, which can reduce non-specific adsorption, thereby meeting the core requirements of the chemiluminescence method "low background-high sensitivity", and solving the problem that the conventional blocking liquid cannot meet the requirements of coupling compatibility and low background.

[0032] 4. The labeling method and application and kit for improving sensitivity and stability of a kit provided by the embodiment of the application utilize SM (PEG)4 activated AP, which has a PEG flexible chain that can form a "double flexible support" with the PEG chain of mPEG-NHS, not only reducing the steric hindrance, but also improving the water solubility of the coupling product and the diffusion efficiency of the chemiluminescence substrate, thereby strengthening the enzyme catalytic luminescence intensity, forming a functional synergy with the chemiluminescence detection, and amplifying the sensitivity advantage.

[0033] 5. The labeling method and kit provided by the embodiment of the present application can provide pg-level sensitivity data (LOD=12.8 pg / mL) by using chemiluminescence method, and has significant advantages in sensitivity (improvement of 92%), stability (12-month activity retention rate of 85.3%), coupling efficiency (91.5%), and the like, and the data is repeatable and verifiable, and is superior to conventional detection methods.

[0034] 6. The labeling method and kit provided by the embodiment of the present application realize the directional labeling of alkaline phosphatase on antibodies, improve the affinity of antibody-alkaline phosphatase complex, and obtain antibody-alkaline phosphatase complex with high affinity, high sensitivity, high specificity and good stability, short process time, simple steps, small risk points in process amplification, controllability, low cost, no need to use dangerous reagents, strong universality and wide application range. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and other related drawings can be obtained by those skilled in the art without creative labor on the premise of not paying creative labor.

[0036] Figure 1 SM(PEG)4 action principle diagram and Traut's reagent action principle diagram provided by the embodiment of the present application;

[0037] Figure 2 Sample correlation diagram of the comparative example 1 of the present application and the identification value;

[0038] Figure 3 Sample correlation diagram of the embodiment 1 of the present application and the identification value;

[0039] Figure 4 Sample correlation diagram of the comparative example 2 of the present application and the identification value. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present application more clear and explicit, the present application will be further described in detail below in combination with embodiments and drawings, and the exemplary embodiments of the present application and the description thereof are only used to explain the present application, and do not limit the present application.

[0041] In the following description, numerous specific details are set forth to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present application.

[0042] Reference throughout this specification to "one embodiment", "an embodiment", "one example", or "an example" means that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment of the application. Thus, appearances of the phrases "in one embodiment", "in an embodiment", "in one example", or "in an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics can be combined in any suitable

[0043] The prior art has the problem that the alkaline phosphatase labeled antibody kit has poor sensitivity and stability, etc. In order to solve the above technical problems:

[0044] In a first aspect, the present application provides a labeling method for improving the sensitivity and stability of a kit, comprising the following steps:

[0045] S1, antibody thiolation: the antibody is treated with a desalting column, the storage solution is replaced with buffer 1, and the raw material concentration is adjusted to 1.25 mg / mL; Traut's reagent is reacted with the antibody in a buffer, and 2-3 free thiol groups per molecule of thiolated antibody are obtained after deprotection with hydroxylamine;

[0046] S2, targeted blocking: the thiolated antibody of step S1 is incubated with a blocking solution containing mPEG-NHS at pH 7.0-7.2 for 20-30 minutes;

[0047] S3, alkaline phosphatase flexible activation: the alkaline phosphatase is first treated with a desalting column, the storage solution is replaced with buffer 2, and a heterobifunctional crosslinking agent is reacted with the alkaline phosphatase in a buffer, and after quenching, an activated alkaline phosphatase containing 3-4 active maleimide groups per molecule is obtained;

[0048] S4, coupling and purification: the blocked thiolated antibody and the activated alkaline phosphatase are incubated in a coupling buffer at 25°C for 2 hours, and gel filtration column purification is performed to obtain an enzyme-labeled antibody.

[0049] In a specific embodiment, in step S1, the Traut's reagent is SATP, which is dissolved in buffer 1 and reacted with the amino group on the surface of the antibody. The reaction time is 0.5-1h, and the reaction temperature is room temperature. After the reaction, the product is stored at 2-8℃ for later use. The molar ratio of SATP to antibody is 10-50:1, and the buffer 1 is a neutral phosphate buffer containing 1% ethylenediaminetetraacetic acid.

[0050] In a specific embodiment, in step S1, the hydroxylamine solution used for deprotection has a final concentration of 50 mM.

[0051] In a specific embodiment, in step S1, the antibody is a murine monoclonal antibody.

[0052] In a specific embodiment, in step S2, the blocking solution includes PBS, EDTA, glycerol, and mPEG-NHS. The specific composition can be as follows: 50 mM PBS, 1 mM EDTA, 5% glycerol, and 5 mM mPEG-NHS.

[0053] In a specific embodiment, in step S3, the heterobifunctional crosslinking agent is an NHS-PEG-maleimide crosslinking agent.

[0054] In a specific embodiment, in step S3, the NHS-PEG-maleimide crosslinking agent is Maleimide-PEG2-NHS, Maleimide-PEG4-NHS, or Maleimide-PEG8-NHS, preferably Maleimide-PEG4-NHS, abbreviated as SM(PEG)4.

[0055] In a specific embodiment, in step S3, the molar ratio of the heterobifunctional crosslinking agent to alkaline phosphatase is 50:1-80:1.

[0056] In a specific embodiment, in step S3, the buffer 2 is a Tris system buffer containing magnesium ions and zinc ions.

[0057] In a specific embodiment, in step S4, the molar ratio of the thiolated antibody to the activated alkaline phosphatase is 10:1-30:1, the reaction temperature is room temperature, and the reaction time is 1h.

[0058] The application uses SATP as an antibody thiolation reagent, can protect the thiol group by acetylation to avoid its oxidation inactivation, then uses mPEG-NHS to specifically close the residual active amino site of the antibody, does not interfere with the subsequent coupling of thiol and maleimide group, the hydrophilicity of PEG chain can reduce non-specific adsorption, improve hydrophilicity, inhibit thiol oxidation, and improve the stability of the labeled product, finally uses the PEG flexible chain of NHS-PEG-maleimide and the PEG chain of mPEG-NHS to form a "double flexible support" cooperatively, not only reduces the steric hindrance, but also improves the water solubility of the conjugate and the diffusion efficiency of the chemiluminescent substrate, further strengthens the enzyme catalytic luminescence intensity, and improves the sensitivity of detection. The principle diagram of SM(PEG)4 and the principle diagram of Traut's reagent are shown in Figure 1

[0059] In the second aspect, the application provides application of the above-mentioned labeling method in chemiluminescence immunoassay.

[0060] In the third aspect, the application provides a chemiluminescence immunoassay kit with high sensitivity and stability, comprising the enzyme-labeled antibody prepared by the above-mentioned labeling method.

[0061] Example 1

[0062] The application embodiment provides a labeling method for improving the sensitivity and stability of the kit, comprising the following steps:

[0063] S1, antibody thiolation

[0064] Antibody pretreatment: take 0.5 mg of anti-IgG monoclonal antibody (concentration 10 mg / mL, volume 50 μL), dilute to 0.4 mL with activation buffer, and ensure the antibody concentration of 1-1.5 mg / mL (optimize the reaction efficiency);

[0065] Thiolation reaction: add 10 mM SATP storage solution 40 μL (antibody: SATP molar ratio = 1:12) to the antibody solution, incubate at room temperature for 30 minutes (acetylated thiol stable combination, no need to strictly avoid light);

[0066] Directed deprotection: add 500 mM hydroxylamine solution 40 μL (final concentration 50 mM), incubate at room temperature for 15 minutes, accurately break the acetyl group, and release the free thiol group (avoid excessive deprotection leading to thiol oxidation);

[0067] ​Preliminary purification: load the reaction solution to a Sephadex G-25 column (equilibrated with coupling buffer), collect the main peak at 280 nm absorption (antibody component) with a volume of about 0.4 mL; determine the sulfhydryl concentration by the DTNB method to ensure that each molecule of antibody contains 2-3 free sulfhydryl groups (optimal coupling ratio);

[0068] S2, targeted blocking

[0069] Add 100 μL of the pre-prepared mPEG-NHS blocking solution working solution to the sulfhydrylated antibody solution of step S1, incubate at room temperature for 30 minutes, and mix gently every 10 minutes;

[0070] S3, alkaline phosphatase flexible activation

[0071] Pre-treatment: take 1.8 mg of AP (divided into 3 groups, 0.6 mg each, concentration 10 mg / mL, volume 60 μL), and dilute each group to 0.4 mL with activation buffer to ensure an AP concentration of 1-2 mg / mL (matching the antibody concentration);

[0072] Flexible activation reaction: add 30 μL of 20 mM SM (PEG)4 stock solution to each group of AP solution drop by drop (AP:SM (PEG)4 molar ratio = 1:18), and incubate at room temperature in the dark for 25 minutes, mixing gently during the incubation;

[0073] Quenching and purification: add 20 μL of 10 mM L-cysteine solution to each group (final concentration 0.5 mM), and incubate at room temperature for 5 minutes to quench the unreacted maleimide groups; load each group of reaction solution separately onto a Sephadex G-25 column (coupling buffer equilibrated), and collect the main peak at 280 nm absorption (activated AP component) with a volume of about 0.4 mL; verify the activation degree by the indirect method of Ellman reagent: each molecule of AP contains 3-4 active maleimide groups;

[0074] S4, coupling and purification

[0075] High-efficiency coupling: mix each group of blocked sulfhydrylated antibody (0.5 mL) with the corresponding group of activated AP (0.4 mL), supplement with coupling buffer to 1 mL, and incubate at 4°C in the dark for 2 hours, mixing gently once every half hour during the incubation (low temperature reduces protein denaturation, and the flexible chain of PEG enhances the efficiency of sulfhydryl-maleimide reaction);

[0076] Final purification: each group of coupling reaction solution was loaded onto a Sephadex G-50 column (equilibrated with a preservation solution), and the first peak (main peak of the conjugate) was collected to remove free small molecule impurities and uncoupled monomers;

[0077] Stable storage: each group of purified conjugate was added to a preservation solution, and was divided into 100 μL / tube and stored at -20°C.

[0078] Comparative Example 1

[0079] The difference from Example 1 is that in step S2, no blocking was performed, and the solution was directly supplemented with a coupling buffer to 0.5 mL, and was left at room temperature for 30 minutes for standby (as a blank control).

[0080] Comparative Example 2

[0081] The difference from Example 1 is that in step S2, 100 μL of a conventional BSA blocking solution working solution was added to the antibody solution, which was incubated at room temperature for 30 minutes, and was gently inverted and mixed every 10 minutes during the incubation (BSA is a conventional blocking agent for immunoassay, which is used to compare the blocking effect).

[0082] Experimental Results

[0083] 1. Coupling efficiency detection (non-reducing SDS-PAGE + gray scale analysis)

[0084] The SDS-PAGE bands of the three groups of conjugates were analyzed by a gel imaging system, and the coupling efficiency (the proportion of the gray scale of the conjugate band to the total gray scale of the bands) was calculated.

[0085] 2. Non-specific adsorption rate detection

[0086] The three groups of conjugates were added to a blank enzyme-labeled plate without coated antigen at the same concentration (0.1 mg / mL), and were incubated at 37°C for 1 hour. After washing, pNPP substrate solution was added, and was incubated at 37°C for 15 minutes. The absorbance at 405 nm (A405) was measured, and the blank substrate solution was used as a control. The non-specific adsorption rate was calculated (adsorption rate = experimental group A405 / blank group A405 x 100%).

[0087] 3. Affinity

[0088] The Ab-AP intermediates of the control group and the experimental group were prepared at the same working concentration of 0.2 μg / mL, and the same gradient samples were measured. The results are shown in Table 1.

[0089] Table 1

[0090] .

[0091] From the above Table 1, in combination with Figures 2-4 It can be seen that, under the same feeding conditions, the reactivity of Example 1 is about twice that of Comparative Example 1, indicating that the phosphatase-antibody affinity of Example 1 is stronger, and theoretically, under the condition of achieving the same reactivity, the antibody consumption of Example 1 can be saved by more than twice.

[0092] 4. Blank limit

[0093] The blank limit reflects the background noise of the detection system to some extent. The smaller the background noise of the detection system, the lower the blank limit, and the more advantageous it is for the detection of low-value samples.

[0094] The calculation formula is: blank limit = M + 2SD; M is the average value; SD is the standard deviation;

[0095] When detecting the blank limit, first adjust the signal value of Example 1 and Comparative Example 1 according to the gradient sample, and the deviation of the signal value of the two groups of reactivity should not exceed 8%, which is used as the final working concentration of each group. Then, 20 blank samples with no antibody to be detected are detected, and then the results are analyzed, as shown in Table 2 below.

[0096] Table 2

[0097] .

[0098] From Table 2, it can be seen that the blank signal value of Example 1 is lower than that of Comparative Example 1, indicating that the alkaline phosphatase-antibody intermediate prepared by the present application can reduce the non-specific adsorption of the reaction system and improve the sensitivity of the reaction system.

[0099] 5. Thermal accelerated stability performance

[0100] The antibody-AP complex labeled by Comparative Example 1, Example 1, and Comparative Example 2 is respectively prepared into reagents, which are respectively placed at 2-8℃ and 37℃ for 7D, and then gradient samples are taken out for determination. The signal retention rate of each group is calculated twice, and the results are shown in Table 3 below.

[0101] Table 3

[0102] .

[0103] From Table 3, it can be seen that the signal retention rate of the reagent stability of Comparative Example 1 is >75%, the signal retention rate of the reagent stability of Example 1 is >92%, and the signal retention rate of the reagent stability of Comparative Example 2 is >83%. It is indicated that the alkaline phosphatase-antibody intermediate prepared by Example 1 has better stability for the reagent kit, which is beneficial to the improvement of product performance.

[0104] 6. Sample correlation

[0105] Gradient positive samples were measured by using Comparative Example 1, Example 1 and Comparative Example 2, respectively, the correlation of the detection results of each group with the standard value of the positive sample was calculated, and the results are shown in Table 4.

[0106] Table 4

[0107] .

[0108] As can be seen from Table 4, the relative deviation and correlation with the standard value of the positive sample, Example 1 is better than Comparative Example 1 and Comparative Example 2. It shows that the intermediate product of alkaline phosphatase-antibody prepared by the present application has obvious improvement in the specificity of the antibody itself, which is beneficial to the improvement of product performance.

[0109] 7. Antibody activity retention rate detection

[0110] The binding activity of the three groups of conjugates with antigens was determined by indirect ELISA method, and the binding activity of the unconjugated original antibody was 100%, and the activity retention rate of the conjugated antibody was calculated, and the results are shown in Table 5.

[0111] Table 5

[0112] .

[0113] The results can be analyzed as follows from Table 5:

[0114] 1. Coupling efficiency: the coupling efficiency (91.5%) of the mPEG-NHS closed group of Example 1 of the present application is significantly higher than that of Comparative Example 1 without closing (65.2%) and Comparative Example 2 closed group (58.9%), because the mPEG-NHS closes the excess amino groups on the surface of the antibody, avoiding the polymerization of the antibody caused by multivalent cross-linking, and at the same time, the hydrophilicity of the PEG chain reduces the hydrophobic aggregation of the antibody and the AP, thereby improving the effective coupling ratio; while the free amino groups of the conventional BSA of Comparative Example 2 will competitively react with the maleimide groups of SM (PEG) 4, thereby reducing the coupling efficiency.

[0115] 2. Non-specific adsorption: the non-specific adsorption rate (9.2%) of Example 1 is much lower than that of Comparative Example 1 and Comparative Example 2, and the core is that the PEG chain of mPEG-NHS forms a hydrophilic shell, shielding the hydrophobic sites on the surface of the antibody, reducing the non-specific binding with the enzyme-labeled plate, thereby reducing the background signal of the kit and indirectly improving the detection sensitivity.

[0116] 3. Sensitivity: LOD (0.007RU / mL) of Example 1 is 75% higher than Comparative Example 1 without blocking and 68% higher than Comparative Example 2 with regular BSA. On one hand, it benefits from the reduction of non-specific adsorption (background signal reduction). On the other hand, the flexible chain of SM (PEG)4 and the PEG chain of mPEG-NHS work together to reduce the steric hindrance between antibody and AP, ensuring the full exposure of antigen binding sites and AP active center, improving the antigen-antibody binding efficiency and enzyme catalytic efficiency.

[0117] 4. Stability: Example 1 performs best in accelerated stability test. The reasons are: (1) the PEG chain of mPEG-NHS wraps the antibody, inhibiting the oxidation of free thiol; (2) ascorbic acid and EDTA in the preservative solution work together to resist oxidation and chelate metal ions; (3) it avoids the risk of degradation of impurities that may be introduced by BSA blocking, while the non-blocking group has the worst stability due to the easy oxidation of thiol and the easy aggregation of protein.

[0118] The materials and reagents used in the examples of the present application are shown in Table 6.

[0119] Table 6

[0120] .

[0121] The above detailed description further illustrates the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A labeling method for improving the sensitivity and stability of a reagent kit, characterized in that, Includes the following steps: S1, Antibody thiolation: The antibody is reacted with Traut's reagent and deprotected by hydroxylamine to obtain thiolated antibody; S2, Targeted Blocking: Incubate the thiolized antibody from step S1 with a blocking solution containing mPEG-NHS; S3, Alkaline phosphatase flexible activation: The alkaline phosphatase is reacted with NHS-PEG-maleimide crosslinking agent and quenched to obtain activated alkaline phosphatase. S4， Conjugation: The blocked thiolized antibody is incubated with activated alkaline phosphatase in conjugation buffer to obtain enzyme-labeled antibody.

2. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S1, the Traut's reagent is SATP, and the molar ratio of SATP to antibody is 10~50:

1.

3. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S1, the antibody is a murine monoclonal antibody.

4. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S2, the blocking solution includes PBS, EDTA, glycerol, and mPEG-NHS.

5. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S3, the NHS-PEG-maleimide crosslinking agent is Maleimide-PEG2-NHS, Maleimide-PEG4-NHS, or Maleimide-PEG8-NHS.

6. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S3, the molar ratio of the NHS-PEG-maleimide crosslinking agent to alkaline phosphatase is 50:1 to 80:

1.

7. The labeling method for improving the sensitivity and stability of the reagent kit according to claim 1, characterized in that, In step S4, the molar ratio of the thiolized antibody to the activated alkaline phosphatase is 10:1 to 30:

1.

8. The application of the labeling method according to any one of claims 1 to 7 in chemiluminescent immunoassay.

9. A chemiluminescent immunoassay kit with high sensitivity and stability, characterized in that, This includes enzyme-labeled antibodies prepared by any of the labeling methods described in claims 1 to 7.

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