Time-resolved immunoassay kit and method for detecting sMIC protein

Abstract

The present invention discloses a time-resolved immunoassay kit and method for detecting sMIC protein. The kit includes sMIC protein standard, sMIC protein capture antibody, lanthanide-labeled detection antibody including first and second detection antibodies, coating solution, blocking solution, enhancement solution, and washing solution, wherein the capture antibody and the detection antibody have different binding epitopes with the MIC protein; the two detection antibodies have different binding activities to different sMIC proteins and the binding activities are complementary. By selecting and selecting two detection antibodies for labeling, the detection sensitivity is improved to the pg level, and the reaction values ​​of the kit to 10 MICAs and 1 MICB are similar, and the detection of more than a dozen substrates can be completed at one time. The use of a fully chemically synthesized inorganic blocking agent, combined with Tween‑20, can not only inhibit nonspecific adsorption, but also enhance the effect of cleaning proteins that are not firmly bound. At the same time, the blocking solution contains a stabilizer component, which will not affect the overall stability of the capture antibody while blocking.

Description

Technical Field

[0001] The present invention relates to the detection field, and in particular to a time-resolved immunoassay kit and method for detecting sMIC protein. Background Art

[0002] Natural Killer (NK) cells recognize infected and tumor cells by scanning changing protein expression patterns through a complex array of receptors expressed on their surface. NK cell receptors can be divided into immunoglobulin-like receptors (such as KIRs, LILRs and NKp30) and C-type lectin-like receptors (such as Ly49s, NKG2D, NKG2 / CD94 and NKp65). NKG2D is one of the most important activating receptors expressed by NK cells in tumor cell recognition.

[0003] The interaction between NKG2D receptors and ligands can activate immune cell-mediated killing. NKG2D ligands are usually not expressed, or expressed at low levels in healthy human tissues, but are usually expressed at high levels in infected cells and cancer cells. In humans, NKG2D ligands include MICA and MICB (class I human major histocompatibility complex-associated proteins A and B). Detecting the levels of MICA and MICB in human tissues can be used as a means of predicting tumor-related diseases. However, there is currently no suitable method for detecting the levels of MICA and MICB in human blood. According to the literature, there are dozens of alleles of MIC protein, among which the MICA genotypes with higher content in the Chinese population are MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, and MICA*045, and their total accounts for more than 90% of the population. The MICA*008 has the highest content and the greatest impact on the human body; the MICB genotypes with higher content are MICB*002 and MICB*005, and their total accounts for more than 70% (http: / / www.allelefrequencies.net / ), and the highest content is MICB*005. This application mainly develops corresponding detection methods for the above MIC protein subtypes.

[0004] Time-resolved fluoroimmunoassay (TRFIA) uses lanthanide elements and their chelates with unique fluorescent properties as tracers to label antigens, antibodies, peptides, nucleic acid probes or biologically active cells. After the reaction system undergoes reactions such as antigen-antibody reaction, bioavidin reaction, nucleic acid probe hybridization, etc., a time-resolved fluorometer is used to measure the fluorescence intensity of the reaction system, thereby further calculating the amount of the target analyte. Compared with other immunoassay techniques, TRFIA has its unique advantages. Many proteins and complexes in serum and biological fluids will produce fluorescence themselves, thereby generating background fluorescence in the measurement. TRFIA is based on the fluorescence decay time of ordinary fluorescent markers, which is about 10 3 -10 6 The lanthanide elements with a maximum of 100 times are used as markers. At the same time, because the Stokes shift of the excitation light and the emission light is large, while the Stokes shift of ordinary fluorescence is only tens of nanometers, the fluorescence intensity of the system is measured after the fluorescence decay of the short-lived fluorescent substance is delayed by time, so as to completely eliminate the background fluorescence. TRFIA has been applied to the clinical detection of various substances, such as the detection of infectious diseases such as hepatitis series and anti-HIV, prenatal screening, sex hormone detection, and the detection of tumor markers such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and tumor-associated antigens (CA50, CA125, etc.).

[0005] TRFIA kits generally include a capture antibody and a labeled detection antibody, which have good specificity. However, there are hundreds of subtypes of sMIC protein, and there are more than 10 subtypes with higher expression frequencies. One detection antibody cannot simultaneously identify more than 10 proteins, resulting in inaccurate test results. TRFIA has not been used to detect sMIC protein in the prior art. Summary of the invention

[0006] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a time-resolved immunoassay kit and method for detecting sMIC protein.

[0007] The technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides:

[0009] A time-resolved immunoassay kit for detecting sMIC protein, comprising sMIC protein standard, sMIC protein capture antibody, lanthanide element labeled detection antibody, coating solution, blocking solution, enhancement solution, and washing solution, wherein:

[0010] The sMIC protein capture antibody and detection antibody have different binding epitopes with the sMIC protein;

[0011] The lanthanide-labeled detection antibody includes a first detection antibody and a second detection antibody, the first detection antibody and the second detection antibody have different binding activities to different sMIC proteins, and the binding activities of the first detection antibody and the second detection antibody to different sMIC proteins are complementary;

[0012] The blocking solution contains 0.5-3 wt.% of a blocking agent, 0.1-1.0 wt.% of mannitol, 0.01-0.2 wt.% of Tween-20, and an appropriate amount of a preservative, and has a pH of 6.0-8.5.

[0013] In some examples of time-resolved immunoassay kits, the enhancement solution contains 0.05-0.3 wt.% TritonX-100, 0.01-0.2 wt.% acetic acid, 0.1-1.0 wt.% β-naphthoyltrifluoroacetone, and 0.5-5 wt.% tri-n-octylphosphine.

[0014] In some examples of the time-resolved immunoassay kit, the lanthanide element is independently selected from at least one of europium Eu, terbium Tb, samarium Sm, and dysprosium Dy.

[0015] In some examples of the time-resolved immunoassay kit, the blocking agent is independently PA1080.

[0016] In some examples of the time-resolved immunoassay kit, the preservative is independently Proclin 300.

[0017] In some examples of the time-resolved immunoassay kit, the mass ratio of the first detection antibody to the second detection antibody is 1:(2-3).

[0018] In some examples of the time-resolved immunoassay kit, the first detection antibody and the second detection antibody are 8B6 and 3F5, respectively.

[0019] In some examples of time-resolved immunoassay kits, the capture antibody is BAMO1.

[0020] In some examples of time-resolved immunoassay kits, the first detection antibody and the second detection antibody are 8B6 and 3F5, respectively, and the capture antibody is BAMO1.

[0021] In some examples of time-resolved immunoassay kits, the molar ratio of the lanthanide chelate to the detection antibody is (30-50):1.

[0022] In some examples of time-resolved immunoassay kits, the coating solution is a PBS solution.

[0023] In some examples of time-resolved immunoassay kits, the washing solution is a PBST solution.

[0024] In some examples of the time-resolved immunoassay kit, the sMIC detected is selected from at least one of MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045 and MICB*005.

[0025] In some examples of the time-resolved immunoassay kit, the sMIC detected is MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045 and MICB*005.

[0026] In some examples of the time-resolved immunoassay kit, the sMIC protein standard is MICA*008 protein.

[0027] The second aspect of the present invention provides:

[0028] A time-resolved immunoassay method for detecting sMIC protein comprises the following steps:

[0029] The sMIC protein capture antibody was diluted with coating solution and coated on the ELISA plate;

[0030] After the ELISA plate is coated, wash the plate with washing solution, add blocking solution, and wash the plate with washing solution after blocking;

[0031] Add the samples and standards to the blocked ELISA plate, and wash the plate with washing solution after sufficient adsorption;

[0032] Add lanthanide-labeled detection antibodies, and wash the plate with washing solution after sufficient reaction;

[0033] Add enhancement solution, stimulate, detect, and determine the content of sMIC protein in the sample;

[0034] The various reagents used therein are as described in the first aspect of the present invention.

[0035] In some examples of time-resolved immunoassay methods, the sMIC protein capture antibody is used at a concentration of 1 to 5 μg / mL.

[0036] In some examples of time-resolved immunoassays, the detection antibody is used at a concentration of 1 to 5 μg / mL.

[0037] In some examples of time-resolved immunoassay methods, the final concentration of lanthanide in each detection well is 0.1-0.5 nmol.

[0038] In some examples of time-resolved immunoassay methods, the first detection antibody has higher binding activity with MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*019, and MICA*027, with detection values ​​not less than 85% of the theoretical added amount, and has lower binding activity with MICA*002, MICA*012, MICA*045, and MICB*005, with detection values ​​less than 85% of the theoretical added amount.

[0039] In some examples of time-resolved immunoassay methods, the second detection antibody has a high binding activity with MICA*002, MICA*012, MICA*045 and MICB*005, and the detection value is not less than 85% of the theoretical addition amount.

[0040] The beneficial effects of the present invention are:

[0041] In some examples of the present invention, the time-resolved immunoassay kit for detecting sMIC protein uses two detection antibodies for labeling, which improves the detection sensitivity to pg level. The reaction values ​​of the kit to 10 MICAs and 1 MICB are similar, and the detection of more than a dozen substrates can be completed at one time.

[0042] The time-resolved immunoassay kit for detecting sMIC protein in some examples of the present invention provides a new method for clinically detecting the content of soluble MICA and soluble MICB in tumor patients. Only an ELISA instrument is needed for detection, and no expensive special instruments are required, which greatly reduces the detection cost.

[0043] The time-resolved immunoassay kit for detecting sMIC protein in some examples of the present invention adopts a fully chemically synthesized inorganic blocking agent, which is used in combination with Tween-20, which can not only inhibit nonspecific adsorption, but also enhance the effect of cleaning weakly bound proteins. At the same time, the blocking solution contains a stabilizer component, which will not affect the overall stability of the capture antibody during blocking. DETAILED DESCRIPTION

[0044] The first aspect of the present invention provides:

[0045] A time-resolved immunoassay kit for detecting sMIC protein, comprising sMIC protein standard, sMIC protein capture antibody, lanthanide element labeled detection antibody, coating solution, blocking solution, enhancement solution, and washing solution, wherein:

[0046] The sMIC protein capture antibody and detection antibody have different binding epitopes with the sMIC protein;

[0047] The lanthanide-labeled detection antibody includes a first detection antibody and a second detection antibody, the first detection antibody and the second detection antibody have different binding activities to different sMIC proteins, and the binding activities of the first detection antibody and the second detection antibody to different sMIC proteins are complementary;

[0048] The blocking solution contains 0.5-3 wt.% of a blocking agent, 0.1-1.0 wt.% of mannitol, 0.01-0.2 wt.% of Tween-20, and an appropriate amount of a preservative, and has a pH of 6.0-8.5.

[0049] Complementary binding activity means that the first detection antibody has relatively strong binding activity to certain types of sMICs in the sMIC to be detected, while the second detection antibody has relatively strong binding activity to the remaining antibodies in the sMIC to be detected.

[0050] In some examples of time-resolved immunoassay kits, the enhancement solution contains 0.05-0.3 wt.% TritonX-100, 0.01-0.2 wt.% acetic acid, 0.1-1.0 wt.% β-naphthoyltrifluoroacetone, and 0.5-5 wt.% tri-n-octylphosphine.

[0051] In some examples of time-resolved immunoassay kits, the lanthanide element is independently selected from at least one of europium Eu, terbium Tb, samarium Sm, and dysprosium Dy. The lanthanide element can be selected accordingly according to specific detection conditions.

[0052] In some examples of time-resolved immunoassay kits, the blocking agent is independently PA1080. PA1080 and Tween-20 are used together to not only inhibit nonspecific adsorption, but also enhance the effect of cleaning weakly bound proteins. At the same time, the blocking solution contains a stabilizer component, which will not affect the overall stability of the capture antibody while blocking.

[0053] The preservative may be any preservative that has no effect or little effect on detection. In some examples of time-resolved immunoassay kits, the preservative is independently Proclin 300.

[0054] In some examples of the time-resolved immunoassay kit, the mass ratio of the first detection antibody to the second detection antibody is 1:(2-3).

[0055] The first detection antibody and the second detection antibody can use commercial detection antibodies. In some examples of time-resolved immunoassay kits, the first detection antibody and the second detection antibody are 8B6 and 3F5, respectively. 8B6 and 3F5 are detection antibodies developed by our company (see CN112574311A, wherein the amino acid sequence of the light chain variable region of 8B6 is DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGLTYLE WYLQKPGQSPKLLIY RVSNRFS GVPDRFSGSGTDFTLKIAEDLGVYYC FQGSHVYRT FGGGTKLEIK, the amino acid sequence of the heavy chain variable region is QVTLRESGPGILQPSQTLSLTCSFSGFSLS TSGVGVG WIRQPSGKGLEWLA HIWGDDDDKRYNPALKG RLTISKDTSNNQVFLKIATVDTTDTATYYCVR MEDL TPYVMEY WGQGTSATVSS; the amino acid sequence of the light chain variable region of 3F5 is DVLMTQIPLSLPVSLGDQASISC RSSQS IVHNNGVTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPRT FGGGTKLEIK, the amino acid sequence of the heavy chain variable region is QVTLKESGPGILQPSQTLSLTCSFSGFSLS TSGMGVG WIRQPSGKGLEWLA HIWWDDDKRYNPALKS RLTISKDTSNNQVFLKIASVDTTDTATYYCVR MEDLTPYVMDY WGQGTSVTVSS), with excellent performance.

[0056] The capture antibody may be a mature commercial capture antibody. In some examples of time-resolved immunoassay kits, the capture antibody is BAMO1. This antibody is a mature capture antibody with stable quality.

[0057] In some examples of time-resolved immunoassay kits, the first detection antibody and the second detection antibody are 8B6 and 3F5, respectively, and the capture antibody is BAMO1.

[0058] In some examples of time-resolved immunoassay kits, the molar ratio of the lanthanide chelate to the detection antibody is (30-50):1.

[0059] The coating solution can be a commonly used coating solution. In some examples of time-resolved immunoassay kits, the coating solution is a PBS solution.

[0060] The washing solution can be a commonly used washing solution. In some examples of time-resolved immunoassay kits, the washing solution is a PBST solution.

[0061] In some examples of the time-resolved immunoassay kit, the sMIC detected is selected from at least one of MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045 and MICB*005.

[0062] In some examples of the time-resolved immunoassay kit, the sMIC detected is MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045 and MICB*005.

[0063] In some examples of the time-resolved immunoassay kit, the sMIC protein standard is MICA*008 protein.

[0064] The second aspect of the present invention provides:

[0065] A time-resolved immunoassay method for detecting sMIC protein comprises the following steps:

[0066] The sMIC protein capture antibody was diluted with coating solution and coated on the ELISA plate;

[0067] After the ELISA plate is coated, wash the plate with washing solution, add blocking solution, and wash the plate with washing solution after blocking;

[0068] Add the samples and standards to the blocked ELISA plate, and wash the plate with washing solution after sufficient adsorption;

[0069] Add lanthanide-labeled detection antibodies, and wash the plate with washing solution after sufficient reaction;

[0070] Add enhancement solution, stimulate, detect, and determine the content of sMIC protein in the sample;

[0071] The various reagents used therein are as described in the first aspect of the present invention.

[0072] In some examples of time-resolved immunoassay methods, the sMIC protein capture antibody is used at a concentration of 1 to 5 μg / mL.

[0073] In some examples of time-resolved immunoassays, the detection antibody is used at a concentration of 1 to 5 μg / mL.

[0074] In some examples of time-resolved immunoassay methods, the final concentration of lanthanide in each detection well is 0.1-0.5 nmol.

[0075] In some examples of time-resolved immunoassay methods, the first detection antibody has higher binding activity with MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*019, and MICA*027, with detection values ​​not less than 85% of the theoretical added amount, and has lower binding activity with MICA*002, MICA*012, MICA*045, and MICB*005, with detection values ​​less than 85% of the theoretical added amount.

[0076] In some examples of time-resolved immunoassay methods, the second detection antibody has a high binding activity with MICA*002, MICA*012, MICA*045 and MICB*005, and the detection value is not less than 85% of the theoretical addition amount.

[0077] The technical solution of the present invention is further explained below in conjunction with examples.

[0078] Embodiment 1:

[0079] BAMO1 was selected as the capture antibody, and europium-labeled 8B6 and 3F5 were used as detection antibodies. The method for europium labeling of 8B6 and 3F5 antibodies is as follows:

[0080] (1) Eu 3+ The molar ratio of chelate to antibody was 50:1. 250 μL antibody (1.67 nmol) was mixed with 12.5 μL Eu 3+ The chelate (84.5 nmol) and 27.5 μL NaHCO3 solution were mixed and incubated at 4°C for 19 h. After incubation, the mixture was separated and purified using Superdex200 prep grade, and 50 mmol / L Tris-HCl (containing 0.9% NaCl, pH 7.8) was passed through the column to collect the solution containing the labeled antibody;

[0081] (2) After diluting the eluent 1000 times with the enhancement solution, the fluorescence intensity of the system was detected using a multifunctional microplate reader. At the same time, the fluorescence intensity of the 1 nmol / L Eu standard solution was used as a reference to calculate the Eu in the eluent. 3+ Determine the OD of the collected protein solution 280nm The value was calculated to calculate the antibody content. Finally, the labeling rate was calculated. The amount of the two antibodies added was adjusted according to the labeling rate so that the amount of europium on the 8B6 antibody was 3 times that on the 3F5 antibody. 3+ High-purity BSA solution was added to the labeled antibody to a final concentration of 0.1% and stored at -20°C.

[0082] Example 2: Effects of different blocking solutions on sMIC detection

[0083] (1) Prepare blocking solutions of different formulas, including 5% BSA solution, 1% PA1080 solution, 1% PA1080 + 0.05% Tween-20 solution, 1% PA1080 + 0.5% mannitol solution, and 1% PA1080 + 0.05% Tween-20 + 0.5% mannitol solution;

[0084] (2) Dilute the capture antibody BAMO1 to an appropriate concentration with 0.01 mol / L PBS (pH 7.2-7.4), add 100 μL to each well, coat the plate on a black 96-well microplate, incubate overnight at 4°C, wash the plate 5 times with washing solution, 300 μL per well, and pat dry; then add 100 μL of blocking solution of different formulations to each well, incubate at 37°C for 2 h, wash the plate 5 times with washing solution, 300 μL per well, and pat dry;

[0085] (3) Add 100 μL of 0.01 mol / L PBS to each well. Prepare and use immediately.

[0086] (4) Incubate at 37°C for 2 h, then wash the plate 5 times with washing buffer, 300 μL per well, and pat dry.

[0087] (5) Add 100 μL of Europium-labeled 8B6 / 3F5 antibody solution to each well and incubate at 37°C for 2 h. Wash the plate 5 times with washing buffer, 300 μL per well, and pat dry.

[0088] (6) Add 200uL of enhancement solution to each well, let it stand at room temperature for 2 minutes, and then measure the fluorescence value on a multi-function microplate reader with an excitation wavelength of 320nm and a detection wavelength of 615nm.

[0089] The measured fluorescence values ​​are shown in Table 1.

[0090] Table 1 Effects of different blocking solutions on test results

[0091]

[0092] The data in Table 1 show that the blank value measured by blocking with the blocking solution of the present invention is lower, indicating that compared with the commonly used blocking agent BSA, the nonspecific adsorption degree is relatively lower after blocking with the blocking solution of the present invention. The optimized blocking solution has a smaller coefficient of variation, more stable measurement results, and a relatively better blocking effect.

[0093] Example 3: Determination of the linear range of the detection kit

[0094] (1) Dilute the capture antibody BAMO1 to an appropriate concentration with 0.01 mol / L PBS (pH 7.2-7.4), add 100 µL to each well, coat the plate on a black 96-well microplate, incubate overnight at 4°C, wash the plate 5 times with washing solution (300 µL per well), pat dry, then add 100 µL of blocking solution to each well and incubate at 37°C for 2 h, wash the plate 5 times with washing solution (300 µL per well), pat dry.

[0095] (2) Add a series of concentration standards, 100 μL / well: Use PBS buffer containing 2.5% BSA to prepare MICA*008 into a series of concentration standard solutions of 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL, and 0 pg / mL. Prepare and use immediately. Incubate at 37°C for 2 h, then wash the plate 5 times with washing solution, 300 μL per well, and pat dry.

[0096] (3) Add 100 μL of 1 μg / mL europium-labeled 8B6 / 3F5 antibody solution to each well (the mass ratio of 8B6 / 3F5 antibody in the solution is 1:3), incubate at 37°C for 2 h, then wash the plate 5 times with washing solution, 300 μL per well, and pat dry.

[0097] (4) Add 200uL of enhancement solution to each well, let it stand at room temperature for 2 minutes, and then measure the fluorescence value on a multi-function microplate reader with an excitation wavelength of 320nm and an emission wavelength of 615nm.

[0098] The experimental results are shown in Table 2.

[0099] Table 2 Determination of linear range

[0100]

[0101] Using logistic curve fitting, we get the equation: y = (A - D) / [1 + (x / C)^B] + D, where A = 7257.92978, B = -0.98803, C = 630.76033, D = 124.17501, R 2 = 0.99920, indicating that the kit has good linearity in the range of 0 to 1000 pg / mL. Based on the mean (M) and standard deviation (SD) of Example 2, the fluorescence value corresponding to M+2SD was obtained, and substituted into the standard curve in this example to calculate the minimum detection limit of the kit to be 5.8 pg / mL.

[0102] Example 4: Effect of different added detection antibodies on fluorescence values

[0103] (1) Dilute the capture antibody BAMO1 to an appropriate concentration with 0.01 mol / L PBS (pH 7.2-7.4), add 100 μL to each well, coat the plate on a black 96-well microplate, incubate overnight at 4°C, wash the plate 5 times with washing solution, 300 μL per well, and pat dry. Then add 100 μL of blocking solution to each well and incubate at 37°C for 2 hours, wash the plate 5 times with washing solution, 300 μL per well, and pat dry.

[0104] (2) Add standard and test samples, 100 μL / well: Use PBS buffer containing 2.5% BSA to prepare MICA*008 into a series of standard solutions with concentrations of 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL, and 0 pg / mL, and prepare MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045, and MICB*005 into 500 pg / mL solutions, and use them immediately. Incubate at 37°C for 2 h, then wash the plate 5 times with washing solution, 300 μL per well, and pat dry;

[0105] (3) Add 100 μL of europium-labeled 1 μg / mL 8B6 / 3F5 antibody solution (8B6 / 3F5 antibody mass ratio in the solution is 1:3) or europium-labeled 1 μg / mL 8B6 antibody solution to different wells, incubate at 37°C for 2 h, then wash the plate 5 times with washing solution, 300 μL per well, and pat dry;

[0106] (4) Add 200uL of enhancement solution to each well, let it stand at room temperature for 2 minutes, and then measure the fluorescence value on a multi-function microplate reader with an excitation wavelength of 320nm and an emission wavelength of 615nm.

[0107] The experimental results are shown in Tables 3 and 4.

[0108] Table 3 Detection results of different sMIC proteins by adding the first detection antibody

[0109]

[0110] Table 4 Detection results of different sMIC proteins by adding two detection antibodies

[0111]

[0112] It can be seen from Tables 3 and 4 that when only one detection antibody is added, the detection recovery rates of MICA*002, MICA*012, MICA*045, and MICB*005 are low and the detection error is large. After adding two detection antibodies and adjusting the ratio of the two, the recovery rates of each sMIC protein tend to be consistent.

[0113] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A time-resolved immunoassay kit for detecting sMIC protein, characterized in that: It includes sMIC protein standard, sMIC protein capture antibody, lanthanide element labeled detection antibody, coating solution, blocking solution, enhancement solution, washing solution, wherein: The sMIC protein capture antibody and detection antibody have different binding epitopes with the sMIC protein; The lanthanide-labeled detection antibody comprises a first detection antibody and a second detection antibody, wherein the first detection antibody and the second detection antibody have different binding activities to different sMIC proteins, and the binding activities of the first detection antibody and the second detection antibody to different sMIC proteins are complementary; wherein the first detection antibody has a higher binding activity to MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*019, and MICA*027, and the detection value is not less than 85% of the theoretical addition amount, and has a lower binding activity to MICA*002, MICA*012, MICA*045, and MICB*005, and the detection value is less than 85% of the theoretical addition amount; the second detection antibody has a higher binding activity to MICA*002, MICA*012, MICA*045, and MICB*005, and the detection value is not less than 85% of the theoretical addition amount; The blocking solution contains 0.5-3 wt.% of a blocking agent, 0.1-1.0 wt.% of mannitol, 0.01-0.2 wt.% of Tween-20, and an appropriate amount of a preservative, and has a pH of 6.0-8.5; The detected sMIC protein is selected from at least one of MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*019, MICA*027, MICA*045 and MICB*005; The first detection antibody and the second detection antibody were 8B6 and 3F5, respectively. The amino acid sequence of the light chain variable region of 8B6 was DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGLTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGTDFTLKIAEDLGVYYCFQGSHVYRTFGGGTKLEIK, and the amino acid sequence of the heavy chain variable region was QVTLRESGPGILQPSQTLSLTCSFSGFSLSTSGVGVGWIRQPSGKGLEWLAHIWGDDDKRYNPALKGRLTISKDTSNNQVFLKIATVDTTDTATYYCVRMEDLTPYVMEYWGQ GTSATVSS; the amino acid sequence of the light chain variable region of 3F5 is DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGVTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK, and the amino acid sequence of the heavy chain variable region is QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSNNQVFLKIASVDTTDTATYYCVRMEDLTPYVMDYWGQGTSVTVSS; The capture antibody is BAMO1.

2. The time-resolved immunoassay kit according to claim 1, characterized in that: The lanthanide element is independently selected from at least one of europium Eu, terbium Tb, samarium Sm, and dysprosium Dy; The sealing agent is independently PA1080; The preservative is independently Proclin 300.

3. The time-resolved immunoassay kit according to claim 1, characterized in that: The mass ratio of the first detection antibody to the second detection antibody is 1:(2-3).

4. The time-resolved immunoassay kit according to claim 1, characterized in that: The molar ratio of the lanthanide chelate to the detection antibody is (30-50):

1.

5. The time-resolved immunoassay kit according to claim 1, characterized in that: The coating solution is PBS solution; The washing solution is PBST solution.

6. The time-resolved immunoassay kit according to any one of claims 1 to 5, characterized in that: The sMIC protein standard is MICA*008 protein.

7. A time-resolved immunoassay method for detecting sMIC protein, characterized in that: The steps include: The sMIC protein capture antibody was diluted with coating solution and coated on the ELISA plate; After the ELISA plate is coated, wash the plate with washing solution, add blocking solution, and wash the plate with washing solution after blocking; Add the samples and standards to the blocked ELISA plate, and wash the plate with washing solution after sufficient adsorption; Add lanthanide-labeled detection antibodies, and wash the plate with washing solution after sufficient reaction; Add enhancement solution, stimulate, detect, and determine the content of sMIC protein in the sample; The various reagents used are as described in any one of claims 1 to 6.

8. The time-resolved immunoassay method according to claim 7, characterized in that: The sMIC protein capture antibody is used at a concentration of 1 to 5 μg / mL; The detection antibody is used at a concentration of 1 to 5 μg / mL, and the final content of the lanthanide element in each detection well is 0.1 to 0.5 nmol.

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