Domain-specific antibodies for detecting mica shed fragments and uses thereof

Abstract

The application relates to the field of biological medicine, and discloses a domain-specific antibody for detecting MICA shedding fragments and application thereof. Two antibodies MICA-16 and MICA-18 of the application can specifically bind to MICA alpha 1 or MICA alpha 2 domains, have strong affinity with MICA alpha 1 or MICA alpha 2, and can accurately and completely recognize MICA shedding fragments; meanwhile, MICA-16 and MICA-18 have no mutual blocking effect, are not combined with MICA alpha 3 domains, do not affect the combination of MICA and MICA alpha 3 antibodies, and also do not affect the combination of NKG2D and MICA. After the two antibodies are used in pairs, the detection sensitivity for MICA shedding fragments alpha 1 or alpha 2 is higher, the two antibodies can be used for detecting MICA shedding fragments in serum, body fluid and cell supernatant, and the detection result can be used as a new clinical reference index for tumor diagnosis and prognosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a domain-specific antibody for detecting MICA detached fragments and its application. Background Technology

[0002] With the development and refinement of modern immunology, the progress of immunotherapy in the field of cancer treatment has attracted much attention. Immunotherapy mainly relies on the body's own immune system to kill tumor cells. Under normal circumstances, the immune system can recognize and kill tumor cells. However, tumor cells can evade the immune system by downregulating the expression of tumor antigens, blocking antigens, and expressing low levels of MHC molecules. Among these, the MICA-NKG2D system on the surface of tumor cells is one of the important pathways for immune cells to recognize and kill cells.

[0003] MICA is a pair of MHCI-class proteins on the surface of tumor cells that mediate signal transduction during cellular stress. The protein structure includes α1, α2, and α3 domains. The distal α1 and α2 domains contain NKG2D binding domains, while the proximal α3 domain contains key antigenic epitopes and MICA hydrolysis detachment sites. MICA was the first discovered NKG2D ligand, binding to NKG2D and activating immune responses. Through the MICA-NKG2D system, it simultaneously regulates both innate and adaptive immune responses, playing a role in immune surveillance against tumorigenesis. Tumor cells, especially those in advanced stages, can hydrolyze MICAα3, releasing MICAα1 and α2, thereby evading immune cell recognition and killing, achieving immune escape. Previous studies have found that blocking the hydrolytic enzyme sites on the MICAα3 domain with antibodies can effectively block the shedding of MICAα1 and α2, thus restoring the NK cell killing ability against tumor cells. It is believed that it is only a matter of time before MICA-related antibody drugs that block the hydrolase sites on the MICAα3 domain are launched on the market.

[0004] On the other hand, studies have found that MICA is present in the serum of patients with malignant tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, and prostate cancer, while normal human tissues and cells generally do not express MICA. Therefore, the MICA content in human serum can serve as a new clinical reference indicator for tumor diagnosis and prognosis. However, there are currently no specific antibodies or corresponding detection kits on the market, either domestically or internationally, targeting the domains of MICA detached fragments (α1 and α2). Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides domain-specific antibodies for detecting MICA detached fragments and their applications. The two antibodies of this invention, MICA-16 and MICA-18, specifically bind to the MICAa1 or MICAα2 domains, enabling accurate and complete recognition of MICA detached fragments. The paired use of these two antibodies provides higher detection sensitivity and can be used to detect MICA detached fragments in serum, body fluids, and cell supernatants. The detection results can serve as a new clinical reference indicator for tumor diagnosis and prognosis.

[0006] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a specific antibody for detecting the MICAα1 domain of the MICA exfoliated fragment, named MICA-16, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2.

[0007] Secondly, the present invention provides a specific antibody for detecting the MICA α2 domain of the MICA exfoliated fragment, named MICA-18, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4.

[0008] Thirdly, the present invention provides the use of MICA-16 and MICA-18 in the preparation of a kit for detecting MICA detached fragments.

[0009] Furthermore, the kit enables quantitative detection.

[0010] Furthermore, the MICA fragment is a MICA detached fragment from serum, body fluid, or cell culture supernatant.

[0011] Fourthly, the present invention provides the use of MICA-16 and MICA-18 in the preparation of kits for diagnosing diseases associated with MICA detached fragments.

[0012] Furthermore, the diseases mentioned include malignant tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, and prostate cancer.

[0013] Furthermore, the MICA fragment is a MICA detached fragment from serum, body fluid, or cell culture supernatant.

[0014] Fifthly, the present invention provides a double-antibody sandwich ELISA kit for detecting MICA detached fragments MICA α1 and MICA α2, using MICA-16 as the capture antibody and biotin-conjugated MICA-18 as the detection antibody; wherein, the MICA-16 comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the MICA-18 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4.

[0015] In a sixth aspect, the present invention provides a double-antibody sandwich ELISA kit for diagnosing diseases associated with MICA α1 and MICA α2 exfoliated fragments, using MICA-16 as a capture antibody and biotin-conjugated MICA-18 as a detection antibody; wherein the amino acid sequence of MICA-16 is as shown in SEQ ID NO: 1. The amino acid sequence of MICA-16 includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region being as shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 2; the amino acid sequence of MICA-18 includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region being as shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 4.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) The sequences of the two antibodies (MICA-16 and MICA-18) provided by this invention are different from those reported in existing literature or patents, and are two novel MICA antibodies.

[0017] (2) The two antibodies provided by this invention can specifically bind to the MICAa1 domain or the MICAα2 domain, and have a strong affinity for the MICA exfoliated fragments α1 or α2 (affinity > 10). 诺 It can accurately and completely identify MICA detached fragments; at the same time, MICA-16 and MICA-18 have no mutual blocking effect, do not bind to the MICAα3 domain, do not affect the binding of MICA to MICAa3 antibody, and do not affect the binding of NKG2D to MICA (that is, MICA-16 and MICA-18 have no competitive inhibitory effect on the binding of MICA to NKG2D).

[0018] (3) When the two antibodies provided by this invention are used in combination, the detection sensitivity for MICA exfoliated fragments α1 or α2 is higher (10-100 pg / mL). Attached Figure Description

[0019] Figure 1 Comparison images of MICA-16 combined with the full-length MICA and MICAα3, respectively; Figure 2 Comparison images of MICA-18 binding with the full length of MICA and MICAα3, respectively; Figure 3 Diagram showing the integration of MICA-NKG2D with MICA-16 and MICA18; Figure 4 The diagram shows the binding of ZSH3#, ZSH4#, and MICA under the blocking conditions of MICA-16 and MICA-18 antibodies. Figure 5 This graph shows the changes in the binding of MICA-18 to the full length of MICA under different concentrations of MICA-16 blocking. Figure 6 Standard curves for the application of MICA-16 and MICA-18 antibodies in ELISA kits. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments.

[0021] General Implementation Examples A specific antibody for detecting the MICAα1 domain of the MICA exfoliated fragment, named MICA-16, comprises a heavy chain amino acid sequence and a light chain amino acid sequence, the light chain amino acid sequence being shown in SEQ ID NO: 1: The heavy chain amino acid sequence is shown in SEQ ID NO: 2.

[0022] A specific antibody for detecting the MICA α2 domain of the MICA exfoliated fragment, named MICA-18, comprises a heavy chain amino acid sequence and a light chain amino acid sequence. The light chain amino acid sequence is shown in SEQ ID NO: 3: DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCKQSYNLFTFGGGTKLEIK. The heavy chain amino acid sequence is shown in SEQ ID NO: 4.

[0023] The use of MICA-16 and MICA-18 in the preparation of kits for detecting MICA detached fragments. Further, the MICA fragments are MICA detached fragments from serum, body fluids, or cell culture supernatants.

[0024] The use of MICA-16 and MICA-18 in the preparation of kits for diagnosing diseases associated with MICA detached fragments. Further, the diseases include malignant tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, and prostate cancer. Further, the MICA fragments are detached MICA fragments from serum, body fluids, or cell culture supernatants.

[0025] A double-antibody sandwich ELISA kit for detecting MICAα1 and MICAα2 detached fragments of MICA, using MICA-16 as the capture antibody and biotin-conjugated MICA-18 as the detection antibody; wherein, MICA-16 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of MICA-18 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4.

[0026] A double-antibody sandwich ELISA kit for diagnosing diseases associated with MICAα1 and MICAα2 exfoliated fragments of MICA, using MICA-16 as the capture antibody and biotin-conjugated MICA-18 as the detection antibody; wherein, MICA-16 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 2; MICA-18 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 4.

[0027] Example 1: Verifying that MICA-16 and MICA-18 bind to the MICAα1 or MICAα2 domains, but not to recombinant proteins containing the MICAα3 domain. Dilute full-length MICA protein and MICAα3 protein to a final concentration of 10 μg / mL using pH=9.6 CBS, add 100 μL to each well, and coat overnight at 4°C. After drying, pre-wash once with TBST washing buffer, and wash four times. Block the enzyme-labeled wells with 10% newborn calf serum TBST and incubate at 37°C for 1 h. Dilute MICA-16 and MICA-18 to 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μg / mL, respectively, and add 100 μL to each well. Incubate at 37℃ for 1 hour; after patting dry, pre-wash once with TBST washing buffer, then wash 4 times; dilute GAM and 10% newborn calf serum TBST at a ratio of 1:3000, add 100 μL to each well, and incubate at 37℃ for 1 hour; after patting dry, pre-wash once with TBST washing buffer, then wash 4 times; add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well, let stand at room temperature for 5 minutes, then add 50 μL of stop solution, set the microplate reader to main wavelength 450 nm, secondary wavelength 630 nm, optical path 1.0, and detect OD value.

[0028] Test results as follows Figure 1 , Figure 2 As shown, MICA-16 and MICA-18 are almost unable to bind to recombinant expressed proteins containing the MICA α3 domain; at the same time, both MICA-16 and MICA-18 can specifically bind to the full-length MICA protein, indicating that the MICA α1 or α2 domains are specifically bound to the antibody.

[0029] Example 2: Verification that MICA-16 and MICA-18 do not competitively inhibit the binding of MICA to NKG2D. Commercial full-length MICA protein was diluted to a final concentration of 10 μg / mL with pH = 9.6 CBS, and 100 μL was added to each well. The plate was coated overnight at 4°C. After drying, the plate was pre-washed once with TBST washing buffer, followed by four washes. The wells were then blocked with 10% newborn calf serum TBST and incubated at 37°C for 1 h. NKG2D was diluted to 50 μg / mL, and a control group was included. After drying the plate, 100 μL was added to each well and incubated at 37°C for 1 h. The washing steps were repeated. Dilute MICA-16 and MICA-18 to 1 μg / mL with 10% newborn calf TBST diluent, add 100 μL to each well, and incubate at 37°C for 1 h; repeat the above washing steps; dilute GAM with 10% newborn calf serum TBST at a ratio of 1:3000, add 100 μL to each well, and incubate at 37°C for 1 h; repeat the above washing steps; add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well, let stand at room temperature for 5 min, then add 50 μL of stop solution, set the microplate reader to main wavelength 450 nm, secondary wavelength 630 nm, optical path 1.0, and detect OD value.

[0030] Test results as follows Figure 3 As shown, there was no significant difference in the reaction between MICA protein and MICA-16 and MICA-18 antibodies before and after binding to NKG2D, indicating that MICA-16 and MICA-18 do not competitively inhibit the binding of MICA to NKG2D.

[0031] Example 3: Verification that the binding of MICA-16 and MICA-18 to MICA does not affect the binding of MICA to MICAα1 and α2 antibodies ZSH3# and 4#. Dilute commercial MICA full-length protein to a final concentration of 5 μg / mL using pH=9.6 CBS, add 100 μL to each well, and coat overnight at 4°C. After drying, pre-wash once with TBST washing buffer, then wash four times. Block the enzyme-labeled wells with 10% newborn calf serum TBST and incubate at 37°C for 1 hour. Dilute MICA-16 and MICA-18 antibodies to 2 μg / mL using 10% newborn calf serum TBST, add 100 μL to each well, and incubate at 37°C for 1 hour. Repeat the above washing steps. Dilute ZSH3# and 4# antibodies with 10% newborn calf serum TBST. Dilute to 1 μg / mL, and set up a blank control group. Add 100 μL to each well and incubate at 37°C for 1 h. Repeat the above washing steps. Dilute GAH and 10% newborn calf serum TBST at a ratio of 1:3000. Add 100 μL to each well and incubate at 37°C for 30 min. After patting dry, pre-wash once with TBST washing buffer and wash 4 times. Add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well, let stand at room temperature for 5 min, and then add 50 μL of stop solution. Set the microplate reader to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a path length of 1.0 to detect the OD value.

[0032] Test results as follows Figure 4 As shown, after MICA protein binds to MICA-16 and MICA-18 antibodies, there is no significant difference in the OD values ​​of ZSH3# and ZSH4# binding to MICA. The binding of MICA-16 and MICA-18 antibodies to ZSH3# and ZSH4# is not competitive and there is no mutual steric hindrance interference.

[0033] Example 4: Verification that MICA-16 and MICA-18 do not block each other. Dilute full-length MICA protein to a final concentration of 10 μg / mL with pH=9.6 CBS, add 100 μL to each well, and coat overnight at 4°C. After drying, pre-wash once with TBST washing buffer, and wash four times. Block the enzyme-labeled wells with 10% newborn calf serum TBST, and incubate at 37°C for 1 h. Dilute MICA-16 antibody to 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μg / mL, and set up blank control wells, adding 100 μL to each well, and incubate at 37°C for 1 h. After drying, pre-wash once with TBST washing buffer, and wash four times. MICA-18 antibody conjugated with biotin and 10 100 μL of 10% newborn calf serum TBST was diluted 1:3000 and added to each well, and incubated at 37°C for 1 h. After drying, the wells were pre-washed once with TBST washing buffer and then washed 4 times. HRP-Streptavidin was diluted 1:4000 with 100 μL of TBST and added to each well, and incubated at 37°C for 30 min. After drying, the wells were pre-washed once with TBST washing buffer and then washed 4 times. 50 μL of chromogenic solution A and 50 μL of chromogenic solution B were added to each well, and the mixture was allowed to stand at room temperature for 5 min. Then, 50 μL of stop solution was added. The microplate reader was set to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a path length of 1.0 to detect the OD value.

[0034] The results are as follows Figure 5 As shown, with the increase of MICA-16 antibody concentration, the OD value of MICA-18 binding to MICA did not change significantly, indicating that MICA-16 has virtually no blocking effect on MICA-18. This suggests that the binding of MICA-16 and MICA-18 antibodies to MICA is non-competitive and there is no mutual steric hindrance interference.

[0035] Example 5: Affinity detection of MICA-16 and MICA-18 Full-length MICA protein was diluted 100-fold and 200-fold with pH=9.6 CBS, 100 μL per well, and coated overnight at 4°C. After drying, the wells were pre-washed once with TBST washing buffer and washed four times. The wells were then blocked with 10% newborn calf serum TBST and incubated at 37°C for 1 hour. MICA-16 and MICA-18 antibodies were diluted 400,000-fold, and then serially diluted 15 times, 100 μL per well, and incubated at 37°C for 1 hour. After drying, the wells were pre-washed once with TBST washing buffer and washed four times. Biotin-conjugated MICA-18 antibody and 10% newborn calf serum TBST were mixed at a ratio of 1:1. 3000 dilution, 100 μL per well, incubated at 37℃ for 1 h; after patting dry, pre-wash once with TBST washing buffer, then wash 4 times; HRP-Streptavidin and 10% newborn calf serum TBST diluted 1:4000, 100 μL per well, incubated at 37℃ for 30 min; after patting dry, pre-wash once with TBST washing buffer, then wash 4 times; add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well, incubate at room temperature for 5 min, then add 50 μL of stop solution. The microplate reader was set to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a path length of 1.0 to detect the OD value. The results are as follows: the affinity of MICA-16 antibody for MICA is approximately 1.53 × 10⁻⁶. 11 The affinity of the MICA-18 antibody for MICA is approximately 2.92 x 10⁻⁶. 10 .

[0036] Table 1. Affinity test results of MICA-16 Table 2. Affinity test results of MICA-18 Example 6: Validation of the detection sensitivity of the MICA-16 and MICA-18 paired ELISA kit Prepare MICA samples of known concentrations and dilute them to linear low values. Detect these samples using a double-antibody sandwich ELISA method with MICA exfoliated fragments. The specific detection method is as follows: (1) Coating: Dilute antibody MICA-16 to 10 μg / mL with CBS solution at pH 9.6, add 100 μL / well to the enzyme-labeled well, seal the membrane and incubate at 4°C overnight; (2) Washing. Discard the liquid in the reaction microwells, pat the plate on a paper towel to absorb the residual liquid, add TBST washing solution, wash the plate 4 times, 1 min each time, and pat dry; (3) Blocking: Add 350 μL of blocking solution to each well of the enzyme-labeled enzyme, incubate at 37°C for 1 h, discard the liquid in the well after the incubation, and pat dry. (4) Sample preparation. Dilute MICA protein with 10% newborn calf TBST to a suitable concentration, and then serially dilute to obtain a standard curve.

[0037] (5) Add sample: Add 100 μL of sample or standard to each of the above-sealed reaction microwells, and use the well with diluent as a blank control. Incubate at 37°C for 1 h. (6) Repeat step 2 for washing; (7) Add biotinylated secondary antibody: Mix biotinylated MICA-18 antibody with 10% newborn calf TBST at a ratio of 1:1000, add 100 μL / well to the enzyme-labeled well, and incubate at 37°C for 30 min. (8) Repeat step 2 for washing; (9) Add HRP-Streptavidin: Dilute HRP-Streptavidin with 10% newborn calf TBST at a ratio of 1:4000, add 100 μL / well to the enzyme-labeled well, and incubate at 37℃ for 1 h; (10) Repeat step 2 for washing; (11) Color development: The TMB color development method was used. The reaction was allowed to stand at room temperature for 10-30 min, and then 50 μL of stop solution was added. The microplate reader was set to a main wavelength of 450 nm, a secondary wavelength of 630 nm, and a light path of 1.0 to detect the OD value.

[0038] (12) Data analysis: Zero the absorbance of the blank well, plot the standard curve with the absorbance of each standard as the ordinate and the corresponding concentration as the abscissa, and calculate the relevant equation Y = aX + b. Substitute the absorbance of each sample into the calculation formula to obtain its corresponding content.

[0039] Test results as follows Figure 6 As shown, MICA-16 and MICA-18 can be paired with each other; with concentration as the X-axis and OD value as the Y-axis, a standard curve with good linearity (r = 0.9966, r > 0.96) y = 0.3247x + 0.1351 can be obtained.

[0040] Table 3 Pairing of MICA-16 and MICA-18 Sensitivity: Using the MICA exfoliated fragment double-antibody sandwich ELISA method described above, blank samples were tested 20 times, and the mean and standard deviation were calculated. The mean plus twice the standard deviation was the limit of detection. The detection results are as follows: the lowest detection sensitivity of this method is approximately 64.9 pg / mL.

[0041] Table 4 Sensitivity Detection After Pairing MICA-16 and MICA-18 Example 7: Application of MICA-16 and MICA-18 in ELISA detection kits Intra-assay precision: Using the above-mentioned MICA exfoliated fragment double antibody sandwich ELISA method, 20 clinical serum samples were tested. The results are as follows: the intra-assay precision of this kit is 8.22%, which meets the testing requirements (the kit requires intra-assay precision CV ≤ 10%).

[0042] Table 4 Intra-batch precision of the reagent kit 1 0.97 2 1.02 3 0.95 4 0.89 5 0.85 6 1.03 7 1.04 8 0.98 9 0.88 10 0.80 11 1.04 12 0.95 13 0.78 14 0.93 15 0.86 16 0.95 17 0.94 18 0.87 19 0.93 20 0.87 mean 0.93 STD 0.08 CV 8.22％ Accuracy: Commercially available MICA eukaryotic protein was diluted to 40, 20, and 10 ng / mL, respectively, and mixed with artificial serum at a ratio of 1:9 to form the experimental groups. An equal volume of 10% newborn calf TBST dilution was mixed with artificial serum at a ratio of 1:9 to form the control group. The above kit was used to detect the experimental and control groups, and the recovery rate was calculated. The results showed that the recovery rates of the experimental group samples with theoretical values ​​of 4, 2, and 1 ng / mL were 95.3%, 102.3%, and 83.2%, respectively, with an average recovery rate of 93.6%, all meeting the detection requirements (the kit requires a recovery rate range of 80%–120%).

[0043] Table 5 Reagent Recovery Rate In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A specific antibody for detecting MICA detached fragments, characterized in that: Named MICA-16, it contains a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO:1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

2. A specific antibody for detecting MICA detached fragments, characterized in that: Named MICA-18, it contains a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

3. The use of the specific antibody as described in claim 1 or 2 in the preparation of a kit for detecting MICA detached fragments.

4. The application as described in claim 3, characterized in that: The MICA detached fragments are MICA detached fragments from body fluids or cell culture supernatants.

5. The use of the specific antibody as described in claim 1 or 2 in the preparation of a kit for diagnosing diseases associated with MICA exfoliated fragments.

6. The application as described in claim 5, characterized in that: The diseases mentioned are lung cancer, colorectal cancer, breast cancer, liver cancer, and prostate cancer.

7. The application as described in claim 5, characterized in that: The MICA detached fragments are MICA detached fragments from body fluids or cell culture supernatants.

8. A double-antibody sandwich ELISA kit for detecting MICA detached fragments MICA α1 and MICA α2, characterized in that: MICA-16 was used as the capture antibody, and biotin-conjugated MICA-18 was used as the detection antibody. MICA-16 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of which is shown in SEQ ID NO:

2. MICA-18 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:3, and the amino acid sequence of which is shown in SEQ ID NO:

4.

9. The reagent kit as described in claim 8, characterized in that: The detection method is a quantitative detection method.

10. A double-antibody sandwich ELISA kit for diagnosing diseases associated with MICA α1 and MICA α2 exfoliated fragments, characterized in that: MICA-16 was used as the capture antibody, and biotin-conjugated MICA-18 was used as the detection antibody. MICA-16 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of which is shown in SEQ ID NO:

2. MICA-18 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:3, and the amino acid sequence of which is shown in SEQ ID NO:4.

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