Human melatonin chemiluminescence immunoassay kit and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro detection, and discloses a human melatonin chemiluminescence immunodetection kit and a preparation method thereof.The human melatonin chemiluminescence immunodetection kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises a magnetic bead coated melatonin antigen, and the reagent R2 comprises a melatonin antibody labeled by a chemiluminescence marker; simple reagent composition is used, expensive instruments and professional personnel are not needed, the cost is reduced, meanwhile, the detection sensitivity is improved, the detection linear range is expanded, and the stability is improved; sample analysis and detection can be automatically carried out through a detection system of a closed kit and instrument, system errors are reduced, errors caused by manual operation are eliminated, the detection efficiency is high, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, and in particular to a human melatonin chemiluminescence immunoassay kit and a preparation method thereof. Background Art

[0002] Melatonin (MT) is one of the hormones secreted by the pineal gland in the brain. Melatonin belongs to the class of indole heterocyclic compounds, and its chemical name is N-acetyl-5-methoxytryptamine, also known as pineal hormone, melatonin, and melatonin. The secretion of melatonin has a distinct circadian rhythm, with secretion suppressed during the day and active secretion at night. Melatonin can inhibit the hypothalamus-pituitary-gonad axis, reduce the levels of gonadotropin-releasing hormone, gonadotropin, luteinizing hormone, and follicle-stimulating hormone, and can directly act on the gonads to reduce the levels of androgens, estrogens, and progesterone. In addition, MT has strong neuroendocrine immunomodulatory activity and free radical scavenging antioxidant ability. Although most people's endogenous melatonin can meet physiological needs, some people may lack the ability to produce appropriate melatonin. At this time, exogenous melatonin supplementation is a relatively safe strategy, so serum MT level is a reference indicator for measuring the level of melatonin in the human body. The serum concentration of melatonin in the human body is lowest at about 4 pm during the day, with an average value of 4.6pg / ml; the serum melatonin level is highest at 4 am, with an average value of 77.5pg / ml.

[0003] At present, the main methods for determining melatonin are enzyme-linked immunosorbent assay and plate chemiluminescence assay. The detection principles of these two methods are basically the same. The antibody is coated on a specific coated plate, and another labeled antibody is used to form a double antibody sandwich structure with the object to be detected, and the relative concentration is calculated by the OD value. The main technical principle of these two detection methods is the double antibody sandwich method, and the sensitivity and linear range are relatively low.

[0004] At present, test kits for melatonin usually use liquid chromatography. For example, patent CN202110651856.5 discloses a test kit for detecting the concentration of serotonin and melatonin in serum. In fact, ultra-high performance liquid chromatography-tandem mass spectrometry is used to detect serotonin and melatonin in serum. The reagents are complex and costly, and expensive instruments are required for use. The operation requires professionals, which is extremely inconvenient.

[0005] Therefore, there is an urgent need to improve the method for determining melatonin, and innovatively propose a human melatonin chemiluminescence immunoassay method and kit, which uses a simple reagent composition and does not require the use of expensive instruments and professional personnel, thereby reducing costs, while improving detection sensitivity, expanding the detection linear range, and improving stability. Summary of the invention

[0006] In order to solve the above existing problems, the present invention discloses a human melatonin chemiluminescence immunoassay kit and a preparation method thereof. The kit is detected by a chemiluminescence method, the reagent components and preparation raw materials in the kit are improved, and microspheres coupled with p-toluenesulfonyl groups are selected to achieve a sensitivity of 1.05 pg / ml, which is suitable for the detection of low-concentration samples; a buffer containing trehalose and tetrasodium iminodisuccinate is used to preserve the melatonin antigen coated with magnetic beads, thereby improving the stability during the storage process, and the kit can be stored at 4°C for up to 2 years without affecting the accuracy and sensitivity of the kit; the closed kit and the instrument detection system can automatically perform sample analysis and detection, thereby reducing system errors and eliminating errors introduced by manual operations, and the detection efficiency is high and the application prospect is broad.

[0007] In one aspect, the present invention provides a human melatonin chemiluminescent immunoassay kit, comprising an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a melatonin antigen coated with magnetic beads, and the R2 reagent comprises a melatonin antibody labeled with a chemiluminescent marker.

[0008] In some embodiments, the magnetic beads of the melatonin antigen coated with magnetic beads are coupled with functional groups, and the functional groups include one or more of carboxyl, amino and toluenesulfonyl (Tosyl).

[0009] Preferably, the magnetic beads of the melatonin antigen coated with magnetic beads are coupled with p-toluenesulfonyl groups.

[0010] From the perspective of principle, the functional group actually affects the connection efficiency between the microspheres and the molecules to be coupled. Different substrates have different effects on the modification of different functional groups of the microspheres. In the present invention, it is obvious that the melatonin antigen is suitable for coating with Tosyl microspheres, which is determined by the molecular structure of melatonin. This is one of the important findings of the present invention.

[0011] In some embodiments, in the melatonin antigen coated with magnetic beads, the mass ratio of the magnetic beads to the melatonin antigen is 1-5:100.

[0012] In some embodiments, the chemiluminescent label comprises one or more of acridinium ester, luminol, isoluminol and terpyridine ruthenium.

[0013] In some embodiments, the molar ratio of the chemiluminescent marker to the melatonin antibody is 5-10:1.

[0014] In some embodiments, the R1 reagent further comprises a first buffer, the magnetic bead-coated melatonin antigen is stored in the first buffer, and the concentration of the magnetic beads is 0.01-1 wt %.

[0015] Preferably, the first buffer comprises 100-500 mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

[0016] Further preferably, the first buffer further comprises trehalose and tetrasodium iminodisuccinate.

[0017] In principle, it is possible that at the microscopic level, the iminodisuccinate ions and trehalose form a network-like arrangement in the solution, which fixes the connecting part of the tosyl microspheres and the melatonin antigen, thereby promoting stability. The specific principle needs to be further demonstrated; the synergistic effect of trehalose and tetrasodium iminodisuccinate on improving the stability of magnetic beads coated with melatonin antigen is another important discovery of the present invention.

[0018] Further research found that the first buffer containing trehalose and tetrasodium iminodisuccinate can be stored at 40°C for 28 days without a significant increase in the number of photons. It can be stored at 4°C for 2 years without affecting the sensitivity and accuracy of the detection. For the concentration ratio of trehalose to tetrasodium iminodisuccinate, after experimental screening, the trehalose content is preferably 0.05-0.5mM, the tetrasodium iminodisuccinate content is preferably 0.02-1mM, and the best is 0.1mM trehalose and 0.1mM tetrasodium iminodisuccinate.

[0019] In some embodiments, the R2 reagent further comprises a second buffer, the melatonin antibody labeled with a chemiluminescent marker is stored in the second buffer, and the concentration of the melatonin antibody labeled with a chemiluminescent marker is 0.1-1 μg / mL.

[0020] Preferably, the second buffer comprises 100-500 mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

[0021] Further preferably, the second buffer further comprises rabbit IgG, trehalose and tetrasodium iminodisuccinate.

[0022] In principle, it may be that at the microscopic level, the iminodisuccinate ions and trehalose form a network-like arrangement in the solution, and the rabbit IgG is distributed in the system in a network-like manner. More binding sites are fully in contact with the interfering antigens in the serum, eliminating nonspecific binding, so that the test value is stably within the acceptable error range of the true value, and the test result is credible; the synergistic effect of rabbit IgG, trehalose and tetrasodium iminodisuccinate on eliminating nonspecific binding in melatonin detection is also an important discovery of the present invention.

[0023] Further studies found that the optimal rabbit IgG content is 0.2-15μg / ml, the optimal trehalose content is 0.05-0.5mM, and the optimal tetrasodium iminodisuccinate content is 0.02-1mM. The optimal content is 5μg / ml rabbit IgG, 0.1mM trehalose, and 0.1mM tetrasodium iminodisuccinate.

[0024] In another aspect, the present invention provides a method for preparing a human melatonin chemiluminescent immunoassay kit, comprising the steps of:

[0025] Step 1, preparation of R1 reagent:

[0026] After the melatonin antigen is replaced, the magnetic beads and the melatonin antigen are suspended overnight, and after adding a blocking agent, the supernatant is collected to obtain 1-5% coated melatonin antigen magnetic beads; then, the magnetic beads are prepared in a buffer solution of 100-500mM Bistris, 0.05-0.1% Tween-20, 0.05-0.1% Proclin300, pH 6.0-8.0 to prepare R1 reagent with a mass ratio of 0.01-1% magnetic bead concentration;

[0027] Step 2, preparation of R2 reagent:

[0028] Putting melatonin binding protein monoclonal antibody into a centrifuge tube, adding carbonate buffer solution after ensuring that the antibody is at the bottom of the centrifuge tube, mixing, adding DMF solution of chemiluminescent marker, and obtaining melatonin binding protein monoclonal antibody labeled with chemiluminescent marker after blocking and purification steps; preparing R2 reagent with a final antibody concentration of 0.1-1 μg / mL by using 100-500 mM Bistris, 0.05-0.1% Tween-20, 0.05-0.1% Proclin300, pH 6.0-8.0 buffer solution for the melatonin binding protein monoclonal antibody concentrated solution;

[0029] Step 3: Combine reagent R1 and reagent R2 to obtain a human melatonin chemiluminescent immunoassay kit.

[0030] In another aspect, the present invention provides a use of p-toluenesulfonyl-modified microspheres for preparing a reagent for improving the sensitivity of human melatonin chemiluminescent immunoassay, wherein the reagent comprises a melatonin antigen coated with p-toluenesulfonyl-modified microspheres.

[0031] In another aspect, the present invention provides a use of a buffer for preparing a reagent for improving the stability of melatonin antigen coated with toluenesulfonyl-modified microspheres, wherein the buffer comprises trehalose and tetrasodium iminodisuccinate.

[0032] In one aspect, the present invention provides a use of a buffer for preparing a reagent for eliminating nonspecific binding between a melatonin antibody labeled with a chemiluminescent marker and a test serum, wherein the buffer comprises rabbit IgG, trehalose and tetrasodium iminodisuccinate.

[0033] In summary, the present invention includes at least one of the following beneficial technical effects:

[0034] 1. The innovative method of using chemiluminescence to detect melatonin uses a simple reagent composition, does not require the use of expensive instruments and professional personnel, reduces costs, and improves the detection sensitivity. The sensitivity is only 1.05pg / ml, which is suitable for the detection of low-concentration samples, expands the detection linear range, and improves stability; the closed test kit and instrument detection system can automatically perform sample analysis and detection, reduce system errors and eliminate errors introduced by manual operations, with high detection efficiency and broad application prospects;

[0035] 2. It was found that the reason why the existing melatonin detection is difficult to detect low-concentration samples is due to the functional groups modified by the microspheres. It was found that tosyl microspheres can significantly improve the detection sensitivity, reducing the detection limit from 4.2pg / ml to 1.05pg / ml;

[0036] 3. Although Tosyl microspheres can improve the detection sensitivity, the coupling is easily disconnected during storage. The present invention finds that the use of a buffer solution including trehalose and tetrasodium iminodisuccinate to store the microsphere-coated melatonin antigen can improve its stability. It can be stored at 4°C for 2 years without affecting the detection accuracy and sensitivity, further expanding the application scope of the present invention;

[0037] 4. The present invention also found that when trehalose and tetrasodium iminodisuccinate are used in combination with rabbit IgG, the nonspecific binding in melatonin detection can be completely eliminated, further increasing the credibility of the detection results of the kit of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Standard curve of human melatonin chemiluminescent immunoassay kit. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials available from commercial sources unless otherwise specified.

[0040] Example 1: Preparation and performance evaluation of a human melatonin chemiluminescent immunoassay kit

[0041] Preparation of R1 reagent: After the melatonin antigen is replaced, the Tosyl magnetic beads and melatonin antigen are suspended overnight, and the blocking agent is added and the supernatant is collected to obtain 2% Tosyl magnetic beads coated with melatonin antigen. The R1 reagent with a magnetic bead concentration of 0.072% is prepared in a buffer solution of 100mM Bistris, 0.05% Tween-20, 0.05% Proclin300, pH 6.0.

[0042] Preparation of R2 reagent: 500 μg of melatonin binding protein monoclonal antibody is placed in a centrifuge tube, and after ensuring that the antibody is at the bottom of the centrifuge tube, a carbonate buffer solution is added, and the mixture is thoroughly mixed. After mixing, acridine ester DMF solution is added, and the molar ratio of melatonin binding protein monoclonal antibody to chemiluminescent marker is 1:10 for labeling, and after blocking, purification and other steps, a melatonin binding protein chemiluminescent marker antibody is obtained. The melatonin chemiluminescent marker antibody concentrated solution is prepared with 100 mM Bistris, 0.05% Tween, 0.05% Proclin300, pH 6.0 buffer to prepare an R2 reagent with a final antibody concentration of 0.5 μg / mL.

[0043] A standard curve was prepared using melatonin protein solutions having calibrator concentrations of 0 pg / mL, 3 pg / mL, 9 pg / mL, 90 pg / mL, 450 pg / mL, 900 pg / mL, and 4500 pg / mL, respectively. The standard curve is shown in the figure below: Figure 1 As shown, the data are shown in Table 1, and the linear correlation coefficient is r=0.9996.

[0044] Table 1

[0045] Concentration (Pg / ml) photon number 0 759674 3 97771 9 37175 90 5730 450 1635 900 960 4500 440

[0046] For analytical sensitivity, the number of photons was measured 20 times for the zero-value calibrator, and the average value was subtracted by twice the standard deviation. The result was inserted into the standard curve of the zero-value calibrator and a similar calibrator to obtain the sensitivity. The sensitivity of the melatonin chemiluminescent immunoassay kit was calculated to be 1.05 pg / mL, and the detection range was 1.05-4500 pg / mL.

[0047] Example 2: Preparation and performance evaluation of a human melatonin chemiluminescent immunoassay kit

[0048] Preparation of R1 reagent: After the melatonin antigen is replaced, the Tosyl magnetic beads and melatonin antigen are suspended overnight, and the blocking agent is added and the supernatant is collected to obtain 3% Tosyl magnetic beads coated with melatonin antigen. The R1 reagent with a magnetic bead concentration of 0.05% is prepared in a buffer solution of 500mM Bistris, 0.1% Tween-20, 0.1% Proclin300, pH 6.5.

[0049] Preparation of R2 reagent: 500 μg of melatonin binding protein monoclonal antibody is placed in a centrifuge tube, and after ensuring that the antibody is at the bottom of the centrifuge tube, a carbonate buffer solution is added, and the mixture is thoroughly mixed. After mixing, acridine ester DMF solution is added, and the molar ratio of melatonin binding protein monoclonal antibody to chemiluminescent marker is 1:5. After blocking, purification and other steps, the melatonin binding protein chemiluminescent marker antibody is obtained. The melatonin chemiluminescent marker antibody concentrated solution is prepared with 500mM Bistris, 0.1% Tween, 0.1% Proclin300, pH6.5 buffer to prepare R2 reagent with a final antibody concentration of 1μg / mL.

[0050] The performance evaluation was carried out according to the method of Example 1. The data are shown in Table 2. The linear correlation coefficient is r=0.9997, the sensitivity is 1.1 pg / mL, and the detection range is 1.1-4500 pg / mL.

[0051] Table 2

[0052] Concentration (Pg / ml) photon number 0 761490 3 95112 9 36789 90 5420 450 1389 900 937 4500 429

[0053] Example 3: Preparation and performance evaluation of a human melatonin chemiluminescent immunoassay kit

[0054] Preparation of R1 reagent: After the melatonin antigen is replaced, the Tosyl magnetic beads and melatonin antigen are suspended overnight, and the blocking agent is added and the supernatant is collected to obtain 3% Tosyl magnetic beads coated with melatonin antigen. The R1 reagent with a magnetic bead concentration of 0.1% is prepared in a buffer solution of 200mM Bistris, 0.08% Tween-20, 0.08% Proclin300, pH8.0.

[0055] Preparation of R2 reagent: 500 μg of melatonin binding protein monoclonal antibody was placed in a centrifuge tube, and after ensuring that the antibody was at the bottom of the centrifuge tube, a carbonate buffer solution was added, and the mixture was thoroughly mixed. After mixing, acridine ester DMF solution was added, and the molar ratio of melatonin binding protein monoclonal antibody to chemiluminescent marker was 1:7. After blocking, purification and other steps, melatonin binding protein chemiluminescent marker antibody was obtained. The melatonin chemiluminescent marker antibody concentrated solution was prepared with 200 mM Bistris, 0.08% Tween, 0.08% Proclin300, pH 8.0 buffer to prepare R2 reagent with a final antibody concentration of 0.75 μg / mL.

[0056] The performance evaluation was performed according to the method of Example 1, with a linear correlation coefficient of r=0.9995, a sensitivity of 1.2 pg / mL, and a detection range of 1.2-4500 pg / mL.

[0057] Table 3

[0058] Concentration (Pg / ml) photon number 0 810216 3 100176 9 38129 90 6136 450 1782 900 1126 4500 654

[0059] Based on Examples 1-3 and other test data during the development of the present invention, the following preferred solutions are finally obtained:

[0060] In the magnetic bead-coated melatonin antigen, the mass ratio of the magnetic beads to the melatonin antigen is 1-5:100.

[0061] The molar ratio of the chemiluminescent marker to the melatonin antibody is 5-10:1.

[0062] The R1 reagent also includes a first buffer, the magnetic bead-coated melatonin antigen is stored in the first buffer, the magnetic bead concentration is 0.01-1wt%; the first buffer includes 100-500mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

[0063] The R2 reagent also includes a second buffer, the melatonin antibody labeled with a chemiluminescent marker is stored in the second buffer, the concentration of the melatonin antibody labeled with a chemiluminescent marker is 0.1-1 μg / mL; the second buffer includes 100-500 mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

[0064] Example 4: Screening of microsphere coupling functional groups

[0065] Generally, magnetic beads are composed of three layers. The innermost layer is polystyrene, the outer layer is a layer of magnetic substance ferroferric oxide, and the outermost layer is a layer of polymer material modified with functional groups, on which different functional groups are coupled.

[0066] The present invention finds that different functional groups have a great influence on the sensitivity of actual detection. In this example, commercially available hydroxyl microspheres, carboxyl microspheres, amino microspheres and toluenesulfonyl (Tosyl) microspheres are used respectively. The preparation and performance analysis of the kit are carried out in the manner of Example 1. The influence of microspheres with different functional groups on the sensitivity is shown in Table 4.

[0067] Table 4: Effect of microspheres with different functional groups on sensitivity

[0068] Microspheres Sensitivity (pg / ml) Hydroxyl Microspheres 4.20 Carboxyl microspheres 2.00 Amino Microspheres 1.80 Tosyl Microspheres 1.05

[0069] It can be seen that different functional groups coupled to the microspheres have a significant effect on the sensitivity. The sensitivity of hydroxyl microspheres is the worst and cannot detect low-concentration samples. Carboxyl microspheres, amino microspheres and Tosyl microspheres are better, among which Tosyl microspheres have the best sensitivity. From the perspective of principle, the functional groups actually affect the connection efficiency between the microspheres and the molecules to be coupled. Different substrates have different effects on the modification of different functional groups of the microspheres. In the present invention, it is obvious that melatonin antigen is suitable for coating with Tosyl microspheres, which is determined by the molecular structure of melatonin. This is one of the important findings of the present invention.

[0070] Example 5: Test of the composition of the first buffer solution

[0071] In Examples 1-3, 100-500mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300 were used as the first buffer to store the magnetic bead-coated melatonin antigen. However, in the subsequent research process, it was found that the magnetic bead-coated melatonin antigen had a certain instability. After testing, the melatonin antigen did not undergo significant denaturation, but when stored at 4°C, the number of photons detected for the same concentration of the sample decreased with the increase in storage time. This is most likely due to the instability of the coupling of Tosyl microspheres and melatonin antigens. Although Tosyl microspheres make the newly prepared R1 reagent have good sensitivity for detection, instability exists during the storage process.

[0072] This example has undergone a large number of experimental screenings and tried a variety of buffers to preserve the magnetic bead-coated melatonin antigen, among which the following three have good effects:

[0073] Reagent 1: 0.1 mM trehalose, 0.1 mM tetrasodium iminodisuccinate, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300;

[0074] Reagent 2: 0.1 wt% water-soluble starch, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin 300;

[0075] Reagent 3: 0.1 mM ascorbic acid, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300.

[0076] In addition, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300 were used as controls. The above-mentioned different buffers were first used to prepare the kit according to the method of Example 1, and then the 100 pg / ml melatonin standard solution was tested, the initial value of the photon number was recorded, and an accelerated test was performed at 40°C. The solution was stored at 40°C for 3 days and 10 days to simulate storage at 4°C for 3 months and 10 months, respectively. Then, the 100 pg / ml melatonin standard solution was tested, the experimental value of the photon number was recorded, and three groups of parallel tests were set to calculate the average value. The results are shown in Table 5.

[0077] Table 5: Effects of different buffer compositions on the stability of magnetic beads coated with melatonin antigen

[0078]

[0079]

[0080] The larger the number of photons, the lower the absorbance of the solid phase carrier (magnetic beads), indicating that the higher the proportion of magnetic beads disconnected from the antigen, the worse the stability. It can be seen that although reagents 2 and 3 have certain good effects, the effects are not significant; while reagent 1 selected trehalose and tetrasodium iminodisuccinate, and the stability was good after being stored at 40°C for 10 days. The present invention has also tested the scheme of adding only trehalose and only adding tetrasodium iminodisuccinate, and there was no positive effect, suggesting that trehalose and tetrasodium iminodisuccinate may have a synergistic effect. From a principle analysis, it may be that at the microscopic level, iminodisuccinate ions and trehalose form a network-like arrangement in the solution, which fixes the connecting part of tosyl microspheres and melatonin antigen, and plays a role in promoting stability. The specific principle needs to be further demonstrated; the synergistic effect of trehalose and tetrasodium iminodisuccinate on improving the stability of magnetic beads coated with melatonin antigen is another important discovery of the present invention.

[0081] Further research found that the first buffer containing trehalose and tetrasodium iminodisuccinate can be stored at 40°C for 28 days without a significant increase in the number of photons. It can be stored at 4°C for 2 years without affecting the sensitivity and accuracy of the detection. For the concentration ratio of trehalose to tetrasodium iminodisuccinate, after experimental screening, the trehalose content is preferably 0.05-0.5mM, the tetrasodium iminodisuccinate content is preferably 0.02-1mM, and the best is 0.1mM trehalose and 0.1mM tetrasodium iminodisuccinate.

[0082] Example 6: Test of the composition of the second buffer

[0083] In Examples 1-3, 100-500mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300 were used as the second buffer to store the melatonin antibody labeled with the chemiluminescent marker. However, in the subsequent research process, it was found that some low-concentration samples were mistakenly detected as high concentrations, indicating that non-specific binding may exist and certain blockers need to be added; common blockers include animal IgG, anti-HAMA monoclonal antibodies, etc., but there is no obvious effect when only the above blockers are added.

[0084] For the convenience of testing, a serum sample with an actual human melatonin concentration of 5.0 μg / ml was taken as a sample to be tested, and a kit was prepared and tested in the manner of Example 1. If the concentration value of the sample to be tested measured by the kit is significantly higher than 5.0 μg / ml outside the error range, non-specific binding is recorded as occurring once. This example has undergone a large number of experimental screenings, and tried a variety of buffers to store the melatonin antibody labeled with a chemiluminescent marker, and then repeated the test 20 times to determine the number of non-specific bindings that occurred in the 20 times. Among them, the following two have good results:

[0085] Reagent 4: 5 μg / ml rabbit IgG, 0.1 mM trehalose, 0.1 mM tetrasodium iminodisuccinate, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300;

[0086] Reagent 5: 5 μg / ml anti-RF factor antibody, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300.

[0087] In addition, 100 mM Bistris, 0.05% Tween-20 and 0.05% Proclin300 were used as controls; the non-specific binding times of the above three groups are shown in Table 6.

[0088] Table 6: Nonspecific binding frequency under different blocking methods (in 20 experiments)

[0089] Reagent 4 Reagent 5 Comparison Nonspecific binding times 0 3 5

[0090] It can be seen that reagent 4 selected rabbit IgG, trehalose and tetrasodium iminodisuccinate to completely block the occurrence of nonspecific binding, stabilize the test value within the error range of the true value, and the test result is credible; the anti-RF factor antibody has a certain effect, but cannot completely block; from the perspective of principle, when trehalose and tetrasodium iminodisuccinate are combined with the passive blocker rabbit IgG, they may also form a similar network structure at the peripheral level, so that the rabbit IgG protein is distributed in a network structure, and when it comes into contact with serum, more binding sites on the surface of rabbit IgG bind to the interfering antigens in the serum, thereby eliminating nonspecific binding. Other principles are not excluded here. The synergistic effect of trehalose and tetrasodium iminodisuccinate combined with the passive blocker rabbit IgG is also an important discovery of the present invention.

[0091] Further studies found that the optimal rabbit IgG content is 0.2-15μg / ml, the optimal trehalose content is 0.05-0.5mM, and the optimal tetrasodium iminodisuccinate content is 0.02-1mM. The optimal content is 5μg / ml rabbit IgG, 0.1mM trehalose, and 0.1mM tetrasodium iminodisuccinate.

[0092] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A human melatonin chemiluminescent immunoassay kit, characterized in that: The method comprises R1 reagent and R2 reagent. R1 reagent comprises melatonin antigen coated with magnetic beads, and R2 reagent comprises melatonin antibody labeled with chemiluminescent marker.

2. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: The magnetic beads of the melatonin antigen coated with the magnetic beads are coupled with functional groups, and the functional groups include one or more of carboxyl groups, amino groups and p-toluenesulfonyl groups.

3. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: In the magnetic bead-coated melatonin antigen, the mass ratio of the magnetic beads to the melatonin antigen is 1-5:

100.

4. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: The chemiluminescent marker includes one or more of acridinium ester, luminol, isoluminol and terpyridine ruthenium.

5. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: The molar ratio of the chemiluminescent marker to the melatonin antibody is 5-10:

1.

6. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: The R1 reagent also includes a first buffer, the melatonin antigen coated with the magnetic beads is stored in the first buffer, and the concentration of the magnetic beads is 0.01-1 wt%.

7. The human melatonin chemiluminescent immunoassay kit according to claim 6, characterized in that: The first buffer comprises 100-500 mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

8. The human melatonin chemiluminescent immunoassay kit according to claim 1, characterized in that: The R2 reagent also includes a second buffer, and the melatonin antibody labeled with the chemiluminescent marker is stored in the second buffer. The concentration of the melatonin antibody labeled with the chemiluminescent marker is 0.1-1 μg / mL.

9. The human melatonin chemiluminescent immunoassay kit according to claim 8, characterized in that: The second buffer comprises 100-500 mM Bistris, 0.05-0.1% Tween-20 and 0.05-0.1% Proclin300.

10. A method for preparing a human melatonin chemiluminescent immunoassay kit, characterized in that: Includes steps: Step 1, preparation of R1 reagent: After the melatonin antigen is replaced, the magnetic beads and the melatonin antigen are suspended overnight, and after adding the blocking agent, the supernatant is collected to obtain 1-5% coated melatonin antigen magnetic beads; then, the R1 reagent with a magnetic bead concentration of 0.01-1wt% is prepared in a buffer solution of 100-500mM Bistris, 0.05-0.1% Tween-20, 0.05-0.1% Proclin300, pH 6.0-8.0; Step 2, preparation of R2 reagent: Putting melatonin binding protein monoclonal antibody into a centrifuge tube, adding carbonate buffer solution after ensuring that the antibody is at the bottom of the centrifuge tube, mixing, adding DMF solution of chemiluminescent marker, and obtaining melatonin binding protein monoclonal antibody labeled with chemiluminescent marker after blocking and purification steps; preparing R2 reagent with a final antibody concentration of 0.1-1 μg / mL by using 100-500 mM Bistris, 0.05-0.1% Tween-20, 0.05-0.1% Proclin300, pH 6.0-8.0 buffer solution for the melatonin binding protein monoclonal antibody concentrated solution; Step 3: Combine reagent R1 and reagent R2 to obtain a human melatonin chemiluminescent immunoassay kit.

Images