A protein antigen combination for detecting acute myocardial infarction and application thereof

Abstract

This invention belongs to the field of biological detection, specifically relating to a protein antigen combination for the detection of acute myocardial infarction and its application. The specific technical solution includes: an antigen combination comprising at least a cTnI (31-164) protein fragment, the amino acid sequence of which is shown in SEQ ID NO: 1. This invention provides an antigen composition for the detection of acute myocardial infarction, exhibiting ideal detection specificity and sensitivity, and can be further prepared into related reagents or kits as needed.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a protein antigen combination and its application for the detection of acute myocardial infarction. Background Technology

[0002] Acute myocardial infarction (AMI) is the most urgent acute coronary syndrome. It is primarily caused by the rupture of atherosclerotic plaques within the coronary arteries, leading to the exposure of a large amount of thrombogenic substances, which subsequently cause acute obstruction of the coronary artery lumen by aggregated platelets, fibrin, and red blood cells. The morbidity and mortality rates of acute myocardial infarction remain high.

[0003] Currently, biochemical testing has become one of the important methods for detecting acute myocardial infarction in clinical practice. Developed biomarkers include serum glutamate-acetyltransferase / aspartate transaminase (SGOT / AST), lactate dehydrogenase (LDH), myoglobin, creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), and troponin. However, the sensitivity and specificity of these biomarkers still need improvement, and the available biomarkers are relatively limited.

[0004] Therefore, expanding the range of biomarkers available and improving the sensitivity and specificity of acute myocardial infarction detection will have enormous application potential. Summary of the Invention

[0005] The purpose of this invention is to provide a protein antigen combination and its application for the detection of acute myocardial infarction.

[0006] To achieve the above-mentioned objective, the technical solution adopted by the present invention is: an antigen combination, wherein the antigen combination includes at least a cTnI (31-164) protein fragment, and the amino acid sequence of the cTnI (31-164) protein fragment is shown in SEQ ID NO: 1.

[0007] Preferably, the antigen combination further includes any one or both of the DCD(20-110) protein fragment and the TNNC1 whole protein, wherein the amino acid sequence of the DCD(20-110) protein fragment is shown in SEQ ID NO: 6, and the amino acid sequence of the TNNC1 whole protein is shown in SEQ ID NO: 3.

[0008] Preferably, the antigen combination includes both a DCD (20-110) protein fragment and the complete TNNC1 protein, and further includes either an HBEGF (19-160) protein fragment or an NPY (29-97) protein fragment; the amino acid sequence of the HBEGF (19-160) protein fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the NPY (29-97) protein fragment is shown in SEQ ID NO: 8. These correspond to combinations 1 and 3 in Table 7 of Example 3, respectively.

[0009] Preferably, the antigen combination includes both DCD (20-110) protein fragments and TNNC1 whole protein, and the antigen combination also includes HSP65 (187-375) protein fragments and HBEGF (19-160) protein fragments; corresponding to combination 6 in Table 6 of Example 3; Alternatively; it also includes: HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment and HBEGF (19-160) protein fragment; corresponding to combination 9 in Table 6 of Example 3; Alternatively; it also includes: HSP65 (187-375) protein fragment, NPPA (26-151) protein fragment and NPY (29-97) protein fragment; corresponding to combination 11 in Table 6 of Example 3; Alternatively; it also includes: HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, NPPA (26-151) protein fragment and NPY (29-97) protein fragment; corresponding to combination 12 in Table 6 of Example 3; Alternatively; it also includes: HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, HBEGF (19-160) protein fragment, NPPA (26-151) protein fragment and NPY (29-97) protein fragment; corresponding to combination 13 in Table 6 of Example 3; The amino acid sequences of the HSP65 (187-375) protein fragment are shown in SEQ ID NO: 5, the amino acid sequences of the HBEGF (19-160) protein fragment are shown in SEQ ID NO: 2, the amino acid sequences of the VEGFR1 (781-1338) protein fragment are shown in SEQ ID NO: 7, the amino acid sequences of the NPPA (26-151) protein fragment are shown in SEQ ID NO: 4, and the amino acid sequences of the NPY (29-97) protein fragment are shown in SEQ ID NO: 8.

[0010] Preferably, the antigen combination includes both cTnI (31-164) protein fragments and DCD (20-110) protein fragments, and also includes HBEGF (19-160) protein fragments and NPY (29-97) protein fragments; corresponding to combination 2 in Table 6 of Example 3; Alternatively; it also includes: HSP65 (187-375) protein fragment, HBEGF (19-160) protein fragment and NPY (29-97) protein fragment; corresponding to combination 7 in Table 6 of Example 3; Alternatively; it also includes: HBEGF (19-160) protein fragment, NPPA (26-151) protein fragment and NPY (29-97) protein fragment; corresponding to combination 8 in Table 6 of Example 3; The amino acid sequence of the HBEGF (19-160) protein fragment is shown in SEQ ID NO: 2, the amino acid sequence of the NPY (29-97) protein fragment is shown in SEQ ID NO: 8, the amino acid sequence of the HSP65 (187-375) protein fragment is shown in SEQ ID NO: 5, and the amino acid sequence of the NPPA (26-151) protein fragment is shown in SEQ ID NO: 4.

[0011] Preferably, the antigen combination includes cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, TNNC1 whole protein and NPY (29-97) protein fragment; corresponding to combination 4 in Table 6 of Example 3; Alternatively; the antigen combination includes cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, TNNC1 whole protein, NPPA (26-151) protein fragment, and NPY (29-97) protein fragment; corresponding to combination 10 in Table 6 of Example 3; The amino acid sequence of the HSP65 (187-375) protein fragment is shown in SEQ ID NO: 5, the amino acid sequence of the TNNC1 whole protein is shown in SEQ ID NO: 3, the amino acid sequence of the NPY (29-97) protein fragment is shown in SEQ ID NO: 8, the amino acid sequence of the VEGFR1 (781-1338) protein fragment is shown in SEQ ID NO: 7, and the amino acid sequence of the NPPA (26-151) protein fragment is shown in SEQ ID NO: 4.

[0012] Accordingly, an antigen combination comprising HSP65 (187-375) protein fragment, TNNC1 protein fragment, DCD (20-110) protein fragment and NPY (29-97) protein fragment; corresponding to combination 5 in Table 6 of Example 3; The amino acid sequence of the HSP65 (187-375) protein fragment is shown in SEQ ID NO: 5, the amino acid sequence of the TNNC1 whole protein is shown in SEQ ID NO: 3, the amino acid sequence of the DCD (20-110) protein fragment is shown in SEQ ID NO: 6, and the amino acid sequence of the NPY (29-97) protein fragment is shown in SEQ ID NO: 8.

[0013] Accordingly, the antigen combination is used in the preparation of products for the detection / identification of acute myocardial infarction.

[0014] Accordingly, reagents or kits for detecting / identifying acute myocardial infarction are prepared using the antigen combination.

[0015] Accordingly, reagents or kits containing the antigen combination for the detection / identification of acute myocardial infarction.

[0016] This invention offers the following beneficial effects: It provides an antigen composition for detecting autoantibodies associated with acute myocardial infarction. This composition can be used to detect the presence of autoantibodies against the aforementioned antigen combination in biological samples from a subject, thereby determining whether the subject has acute myocardial infarction. Using the antigen composition provided by this invention, it is possible to predict whether a subject has a risk of developing or recurring acute myocardial infarction. It can be used to detect myocardial infarction and chronic myocardial damage (such as myocardial loss caused by tumor drugs), and can also be used to assist in the identification of coronary heart disease and guide the use of clinical drugs. The biological sample can be serum, plasma, whole blood, saliva, oral mucosal swabs, urine, lymph, cerebrospinal fluid, etc. Depending on the specific circumstances, the biological sample can be pretreated by extraction, dilution, enrichment, etc., making the methods of use diverse and easy to operate. Detailed Implementation

[0017] This invention screened and obtained three protein fragment antigens closely related to acute myocardial infarction: cTnI (31-164), HSP65 (187-375), and VEGFR1 (781-1338). These three protein fragments can be used alone, in combination, or in combination with other biomarkers to prepare products (e.g., kits) for the detection and diagnosis of acute myocardial infarction.

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0019] Example 1: Construction, expression, and purification of recombinant vectors for antigens One possible method for obtaining the antigen is to synthesize the encoding DNA of the protein antigen, use the synthesized DNA as a template, design primers, and clone the gene fragment of the protein antigen or its fragment into an expression plasmid using molecular cloning techniques such as PCR, enzyme digestion, and ligation. The protein is then expressed in *E. coli*, yeast, or cells, and subsequently purified by chromatography to obtain the target protein. Additionally, tags such as Trx, GST, AVI, HIS, and c-myc can be selectively added to the protein antigen or its fragment. Adding these tags facilitates the purification or labeling of the protein antigen but does not fundamentally alter the binding characteristics between the antigen and its own antibody.

[0020] The proteins that are potentially associated with the detection of acute myocardial infarction were selected, and their respective database IDs are shown in Table 1.

[0021] Table 1. Database ID Comparison Table for Potential Proteins

[0022] Using a human cDNA library (purchased from Invitrogen) or fully synthesized DNA as templates, primers were designed, and the protein gene fragment was cloned into the pET28 plasmid using molecular cloning techniques such as PCR, enzyme digestion, and ligation. Simultaneously, HIS and FLAG tags were added to the N-terminus of the protein to form a fusion protein. The resulting recombinant expression vector was identified by DNA sequencing to confirm the presence of the correct protein gene fragment. It should be noted that the tagging is only for ease of identification and extraction and does not decisively affect the protein's function as an antigen. When using this method, it is acceptable to omit the tag, add other tags as needed, or employ other labeling / identification methods.

[0023] The recombinant plasmid containing the protein gene fragment was transformed into *E. coli* BL21(DE3) competent cells. Clones were picked and inoculated into LB medium and cultured in a shaker at 37°C. When the bacterial density OD... 600 When the temperature reached approximately 0.8, the temperature was lowered to 16°C, and 0.1 mM isopropyl thio-β-D-galactoside (IPTG) was added to each LB medium to induce expression overnight, thus obtaining bacterial cells.

[0024] The induced bacterial cells were collected by centrifugation and washed twice with PBS. The cells were resuspended and dispersed with lysis buffer (5-10 mL per g of cells), placed on ice, and sonicated (200 W, 5 s for lysis, 5 s rest). After lysis, the cells were centrifuged at 13000 rpm and 10°C for 20 minutes. The supernatant was collected and purified by Ni column affinity chromatography and molecular sieve chromatography. SDS-PAGE electrophoresis was used to confirm the molecular weight and purity of the proteins. The concentration was determined using the Bradford method, and the purified proteins were stored at -80°C for later use. The purified candidate proteins were thus obtained. Specifically, the whole proteins of cTnI, TNNC1, and HSP65 were obtained; HBEGF (19-160), NPPA (26-151), DCD (20-110), and NPY (29-97) protein fragments were obtained; the whole VEGFR1 protein was not obtained.

[0025] Example 2: Demonstration of the effectiveness of individual candidate proteins in detecting acute myocardial infarction samples 1. The solutions and reagents used in this embodiment are as follows: (1) The coating buffer is PBS buffer with pH=7.4. The preparation method is as follows: accurately weigh 3.58g Na2HPO4·12H2O, 0.23g KH2PO4·2H2O, 0.2g KCl and 8.0g NaCl, dissolve them in water, and add water to make up to 1L.

[0026] (2) Blocking buffer / sample diluent / antibody diluent: Dissolve 10g BSA (bovine serum albumin) in 800ml coating buffer and bring the volume up to 1L with coating buffer.

[0027] (3) Washing solution: Prepare fresh before use. Before use, add 0.5% Tween20 (V / V) to the coating buffer, pH=7.4.

[0028] (4) TMB colorimetric reagent, purchased from KPL Company.

[0029] (5) Termination solution: 1M hydrochloric acid.

[0030] 2. Solid-phase coating of the test proteins. Dilute each purified candidate protein obtained in Example 1 to 5 μg / mL with coating buffer, add 50 μL to each well of a 96-well plate, and coat overnight at 4°C. The next day, discard the solution, spin dry, and wash three times with 200 μL of washing buffer each time. Then add 200 μL of blocking buffer to each well, incubate at room temperature for 1 hour, discard the blocking buffer, spin dry, and wash three more times with 200 μL of washing buffer each time, then spin dry again; obtain the solid-phase coated antigen in the 96-well plate.

[0031] 3. Add the test sample. Dilute the human serum sample 100-fold with the sample diluent, and add 50 μL of the diluted test sample to each well of the 96-well plate containing the test protein. Then place the 96-well plate on a microplate shaker and incubate at room temperature for 1 hour; spin dry, wash three times with washing buffer, 200 μL per well each time, and spin dry again.

[0032] 4. Add enzyme-labeled secondary antibody. Dilute 1.0 mg / mL horseradish peroxidase-labeled recombinant goat anti-human immunoglobulin G antibody (purchased from Jackson ImmunoResearch Inc.) 20,000 times with antibody dilution buffer, and add 50 μL to each well of the 96-well plate treated in step 3. Then place the 96-well plate on a microplate shaker and incubate at room temperature for 0.5 h. Shake the plate dry, wash three times with washing buffer, 200 μL per well each time, and shake dry again.

[0033] 5. Colorimetric reaction and optical density reading. In the 96-well plate after step 4, add 50 μL of TMB colorimetric reagent to each well, shake for 15 s, react at room temperature in the dark for 15 min, and then add 50 μL of stop solution; then use a microplate reader to read the absorbance value at a wavelength of 450 nm to obtain the detection signal (S) of each sample.

[0034] 6. Sensitivity and Specificity Analysis. 384 positive samples (serum from patients diagnosed with myocardial infarction) and 384 negative samples (serum from healthy subjects) were collected. The detection signal (S) of each sample was measured using the method described above (absorbance at 450 nm wavelength). Using the negative samples as negative reference samples, the mean (M) and standard deviation (SD) of the detection signals (S) of all negative reference samples were calculated, with M+3SD as the cut-off value. Samples with a detection signal (S) ≥ the cut-off value (S ≥ M+3SD) were defined as positive; samples with a detection signal (S) < the cut-off value (S < M+3SD) were defined as negative.

[0035] Specificity and sensitivity were calculated based on positive and negative sample results. Specificity refers to the proportion of healthy subject samples correctly identified as negative, calculated as the number of negative samples identified as negative divided by the total number of negative samples. Sensitivity refers to the proportion of enrolled positive patient samples identified as positive, calculated as the number of positive samples identified as positive divided by the total number of positive samples. The sensitivity and specificity were calculated when each tested protein was used as an antigen for sample testing. The results are shown in Table 2. " / " indicates that the corresponding protein was not obtained and was not measured.

[0036] Table 2 Comparison of detection results for each candidate protein

[0037] The results showed that most of the potential proteins or protein fragments had low sensitivity in detecting acute myocardial infarction when used alone. Although HSP65 had relatively higher sensitivity, its low specificity led to a high false positive rate, making its clinical application difficult.

[0038] 7. The amino acid sequences and structures of the whole proteins of cTnI, HSP65, and VEGFR1 were analyzed. After extensive preliminary experiments, different sequence fragments were selected, and the sensitivity and specificity for detecting acute myocardial infarction were determined according to the method in step 6. The selected sequence fragments and the detection results are shown in Table 3.

[0039] Table 3 Comparison of Protein Fragments and Detection Results

[0040] The results showed that compared with whole protein and other protein fragments, cTnI (31-164) exhibited significantly improved sensitivity while maintaining high specificity. Although the sensitivity of HSP65 (187-375) was slightly lower than that of HSP65 whole protein and HSP65 (1-540), its specificity was significantly improved, reducing the possibility of false positives while ensuring detection rate. Therefore, HSP65 (187-375) was selected as the preferred fragment. The specificity results of VEGFR1 (781-1338) and VEGFR1 (27-242) were comparable, with little difference in sensitivity values. However, VEGFR1 (781-1338) had a larger AUC area, which could reduce the possibility of false positives. Therefore, VEGFR1 (781-1338) was selected as the preferred fragment.

[0041] 8. Using the protein fragments listed in Table 3, an indirect enzyme-linked immunosorbent assay (ELISA) was performed on 96 patients diagnosed with coronary heart disease (CHD) and 96 healthy controls of similar age and sex. The CHD patients included 50 men and 46 women, aged 40-79 years, with a mean age of 54 years. The diagnostic methods used for confirming the diagnosis met the World Health Organization (WHO) clinical diagnostic criteria for CHD. Samples were collected from CHD patients immediately upon definitive diagnosis.

[0042] The results showed that, compared with the serum response of normal individuals, antibodies produced by cTnI (31-164), HSP65 (187-375), and VEGFR1 (781-1338) elicited more pronounced antigen-antibody reactions in serum (manifested as significantly elevated OD values) compared to whole protein and other protein fragments. This demonstrates the potential of these protein fragments for screening and detecting coronary heart disease.

[0043] Based on steps 6-8, the final selected candidate proteins (fragments) are shown in Table 4.

[0044] Table 4. Comparison of Candidate Proteins (Fragments) and Detection Results

[0045] Example 3: Demonstration of the effect of protein composition combined with detection of acute myocardial infarction samples 1. Use the "dual indicator" method, which involves detecting two protein antigens.

[0046] The "dual-index" method refers to the following: when a sample is tested using a certain antigen combination, if both antigens in the combination produce positive detection signals, the sample is considered positive; otherwise, it is considered negative. The remaining procedures and the definitions of sensitivity and specificity are the same as in Example 2. The results are shown in Table 5.

[0047] Table 5 Comparison of Results of Double Antigen Combination Detection

[0048] The results showed that the "dual-indicator" method, using dual-antigen combinations to detect acute myocardial infarction, had extremely high specificity and could largely avoid false positives, but the positive detection rate was low and the sensitivity was not ideal. Among them, combinations 2, 6, 9, 17, 22, and 25 had relatively higher sensitivity, and these combinations were selected for further experiments.

[0049] 3. Add proteins to the antigen combinations. Using the "dual-indicator" method from step 2 (in this step, a positive result is determined by two or more positive antigen detection signals, otherwise a negative result), and employing the sample and sensitivity / specificity assay methods from Example 2, the specificity and sensitivity of the antigen combinations were tested. The results are shown in Table 6. It should be noted that the inventors obtained the antigen combinations shown in Table 6 after extensive preliminary experiments, and did not only test the combinations listed in Table 6. Due to space limitations, only some of the more effective combinations are selected here.

[0050] Table 6 Comparison of Antigen Combination Detection Results for Acute Myocardial Infarction

[0051] The results showed that combinations 1-13 could significantly improve the detection rate and sensitivity while maintaining high specificity, thus effectively enhancing their clinical application value.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. An antigen combination, characterized in that: The antigen combination consists of cTnI (31-164) protein fragments, HBEGF (19-160) protein fragments, DCD (20-110) protein fragments, and the complete TNNC1 protein; or; The antigen combination consists of cTnI (31-164) protein fragments, HBEGF (19-160) protein fragments, DCD (20-110) protein fragments, and NPY (29-97) protein fragments; or; The antigen combination consists of cTnI (31-164) protein fragments, DCD (20-110) protein fragments, TNNC1 whole protein, and NPY (29-97) protein fragments; or; The antigen combination consists of cTnI (31-164) protein fragments, HSP65 (187-375) protein fragments, TNNC1 whole protein, and NPY (29-97) protein fragments; or; The antigen combination consists of cTnI (31-164) protein fragments, HSP65 (187-375) protein fragments, HBEGF (19-160) protein fragments, TNNC1 whole protein, and DCD (20-110) protein fragments; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, HBEGF (19-160) protein fragment, DCD (20-110) protein fragment and NPY (29-97) protein fragment; or; The antigen combination consists of cTnI (31-164) protein fragments, HBEGF (19-160) protein fragments, NPPA (26-151) protein fragments, DCD (20-110) protein fragments, and NPY (29-97) protein fragments; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, HBEGF (19-160) protein fragment, TNNC1 whole protein, and DCD (20-110) protein fragment; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, TNNC1 whole protein, NPPA (26-151) protein fragment, and NPY (29-97) protein fragment; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, TNNC1 whole protein, NPPA (26-151) protein fragment, DCD (20-110) protein fragment and NPY (29-97) protein fragment; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, TNNC1 whole protein, NPPA (26-151) protein fragment, DCD (20-110) protein fragment, and NPY (29-97) protein fragment; or; The antigen combination consists of cTnI (31-164) protein fragment, HSP65 (187-375) protein fragment, VEGFR1 (781-1338) protein fragment, HBEGF (19-160) protein fragment, TNNC1 whole protein, NPPA (26-151) protein fragment, DCD (20-110) protein fragment and NPY (29-97) protein fragment; The amino acid sequences of the cTnI (31-164) protein fragment are shown in SEQ ID NO: 1, the amino acid sequences of the HBEGF (19-160) protein fragment are shown in SEQ ID NO: 2, the amino acid sequences of the TNNC1 whole protein are shown in SEQ ID NO: 3, the amino acid sequences of the NPPA (26-151) protein fragment are shown in SEQ ID NO: 4, the amino acid sequences of the HSP65 (187-375) protein fragment are shown in SEQ ID NO: 5, the amino acid sequences of the DCD (20-110) protein fragment are shown in SEQ ID NO: 6, the amino acid sequences of the VEGFR1 (781-1338) protein fragment are shown in SEQ ID NO: 7, and the amino acid sequences of the NPY (29-97) protein fragment are shown in SEQ ID NO:

8.

2. The use of the antigen combination described in claim 1 in the preparation of products for detecting / identifying acute myocardial infarction.

3. A reagent or kit for detecting / identifying acute myocardial infarction prepared using the antigen combination described in claim 1.

4. A reagent or kit for detecting / identifying acute myocardial infarction comprising the antigen combination of claim 1.

Images