Protein antigen combination for acute myocardial infarction detection, and use thereof

Abstract

Provided are a protein antigen combination for acute myocardial infarction detection, and the use thereof. The antigen combination at least comprises a cTnI (31-164) protein fragment having an amino acid sequence as shown in SEQ ID NO: 1. The antigen combination can be used in an antigen composition for detecting acute myocardial infarction, and can be further prepared into a related reagent or kit on the basis of need.

Description

A protein antigen combination for acute myocardial infarction detection and application TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a protein antigen combination for acute myocardial infarction detection and application. BACKGROUND

[0002] Acute myocardial infarction (AMI) is the most urgent acute coronary syndrome. It is mainly caused by the rupture of the coronary atherosclerotic plaque, which leads to the exposure of a large amount of thrombogenic material, and then the acute occlusion of the coronary lumen by aggregated platelets, fibrin and red blood cells. The morbidity and mortality of acute myocardial infarction are high.

[0003] At present, biochemical detection has become one of the important means for detecting acute myocardial infarction in clinical practice. The developed biomarkers include serum glutamic oxalacetic transaminase / aspartate aminotransferase (SGOT / AST), lactate dehydrogenase (LDH), myoglobin, creatine kinase (CK), creatine kinase MB isozyme (CK-MB) and troponin (Tropoin) and the like. However, the sensitivity and specificity of these markers in detection need to be improved, and the available biomarkers are limited.

[0004] Therefore, if the available range of biomarkers can be expanded and the sensitivity and specificity of acute myocardial infarction detection can be improved, it will have great application prospects. SUMMARY

[0005] The present application aims to provide a protein antigen combination for acute myocardial infarction detection and application.

[0006] To achieve the above-mentioned application purposes, the technical solution adopted by the present application is as follows: an antigen combination, which at least comprises 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 comprises any one or both of a DCD (20-110) protein fragment and a TNNC1 whole protein, 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 simultaneously comprises the DCD(20-110) protein fragment and the TNNC1 full protein, and further simultaneously comprises: the HBEGF(19-160) protein fragment or the NPY(29-97) protein fragment; the amino acid sequence of the HBEGF(19-160) protein fragment is shown as SEQ ID NO: 2, and the amino acid sequence of the NPY(29-97) protein fragment is shown as SEQ ID NO: 8. Corresponding to combination 1 and combination 3 of Table 7 in Example 3, respectively.

[0009] Preferably, the antigen combination simultaneously comprises the DCD(20-110) protein fragment and the TNNC1 full protein, and further simultaneously comprises: the HSP65(187-375) protein fragment and the HBEGF(19-160) protein fragment; corresponding to combination 6 of Table 6 in Example 3; or further simultaneously comprises: the HSP65(187-375) protein fragment, the VEGFR1(781-1338) protein fragment and the HBEGF(19-160) protein fragment; corresponding to combination 9 of Table 6 in Example 3; or further simultaneously comprises: the HSP65(187-375) protein fragment, the NPPA(26-151) protein fragment and the NPY(29-97) protein fragment; corresponding to combination 11 of Table 6 in Example 3; or further simultaneously comprises: the HSP65(187-375) protein fragment, the VEGFR1(781-1338) protein fragment, the NPPA(26-151) protein fragment and the NPY(29-97) protein fragment; corresponding to combination 12 of Table 6 in Example 3; or further simultaneously comprises: the HSP65(187-375) protein fragment, the VEGFR1(781-1338) protein fragment, the HBEGF(19-160) protein fragment, the NPPA(26-151) protein fragment and the NPY(29-97) protein fragment; corresponding to combination 13 of Table 6 in Example 3; the amino acid sequence of the HSP65(187-375) protein fragment is shown as SEQ ID NO: 5, the amino acid sequence of the HBEGF(19-160) protein fragment is shown as SEQ ID NO: 2, the amino acid sequence of the VEGFR1(781-1338) protein fragment is shown as SEQ ID NO: 7, the amino acid sequence of the NPPA(26-151) protein fragment is shown as SEQ ID NO: 4, and the amino acid sequence of the NPY(29-97) protein fragment is shown as SEQ ID NO: 8.

[0010] Preferably, the antigen combination comprises both the cTnl (31-164) protein fragment and the DCD (20-110) protein fragment, and further comprises: the HBEGF (19-160) protein fragment and the NPY (29-97) protein fragment; corresponding to Combination 2 of Table 6 of Example III; or further comprises: the HSP65 (187-375) protein fragment, the HBEGF (19-160) protein fragment and the NPY (29-97) protein fragment; corresponding to Combination 7 of Table 6 of Example III; or further comprises: the HBEGF (19-160) protein fragment, the NPPA (26-151) protein fragment and the NPY (29-97) protein fragment; corresponding to Combination 8 of Table 6 of Example III; the amino acid sequence of the HBEGF (19-160) protein fragment is shown as SEQ ID NO: 2, the amino acid sequence of the NPY (29-97) protein fragment is shown as SEQ ID NO: 8, the amino acid sequence of the HSP65 (187-375) protein fragment is shown as SEQ ID NO: 5, and the amino acid sequence of the NPPA (26-151) protein fragment is shown as SEQ ID NO: 4.

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

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

[0013] Correspondingly, the antigen combination is used for preparing a product for detecting / identifying acute myocardial infarction.

[0014] Correspondingly, a reagent or kit for detecting / identifying acute myocardial infarction prepared by using the antigen combination.

[0015] Correspondingly, a reagent or kit for detecting / identifying acute myocardial infarction comprising the antigen combination.

[0016] The present application has the following beneficial effects: the present application provides an antigen combination for detecting acute myocardial infarction related autoantibodies, which can be used to detect whether there is an autoantibody against the above-mentioned antigen combination in a biological sample from a subject, so as to determine whether the subject has acute myocardial infarction. By using the antigen combination provided by the present application, it can be predicted whether the subject has a risk of suffering from acute myocardial infarction or a risk of recurrence of acute myocardial infarction, which can be used to detect myocardial infarction and chronic myocardial damage (such as myocardial damage caused by tumor drugs), and can also be used to assist in identifying coronary heart disease and guiding the use of clinical drugs. The biological sample can be serum, plasma, whole blood, saliva, oral mucosa swab, urine, lymph, cerebrospinal fluid, etc. According to the specific circumstances, the biological sample can be pretreated by extraction, dilution, enrichment, etc. The method is various and easy to operate. DETAILED DESCRIPTION

[0017] The present application screens and obtains three protein fragment antigens closely related to acute myocardial infarction: cTnI(31-164), HSP65(187-375) and VEGFR1(781-1338). The above-mentioned three protein fragments can be used alone or in combination or in combination with other biomarkers to prepare a product (such as a kit) for detecting and diagnosing acute myocardial infarction.

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. If not specifically indicated, the technical means used in the embodiments are conventional means familiar to those skilled in the art. The obtained data are all average values obtained after at least three repetitions, and the data obtained in each repetition are all valid data.

[0019] Example 1: Construction, expression and purification of recombinant vector of antigen The method for obtaining the antigen can be selected as follows: synthesizing the coding DNA of the protein antigen, designing primers, cloning the gene fragment of the protein antigen or the fragment thereof to an expression plasmid through PCR, enzyme digestion, ligation and other molecular cloning means, then expressing through E. coli, yeast or cells, and then purifying the target protein through chromatography. Meanwhile, a Trx, GST, AVI, HIS, c-myc and the like can be selectively added to the protein antigen or the fragment thereof. Adding these tags can facilitate the purification or labeling of the protein antigen, but will not change the binding properties of the antigen and its own antibody in essence.

[0020] The potential proteins related to detection of acute myocardial infarction are selected, and the database IDs of the proteins are shown in Table 1.

[0021] Table 1: Database ID of each potential protein

[0022] The primers are designed respectively by taking the human cDNA library (purchased from Invitrogen Corporation) or the full gene synthetic DNA as a template, and the gene fragment of the protein is cloned to the pET28 plasmid through PCR, enzyme digestion, ligation and other molecular cloning means. Meanwhile, a HIS, FLAG and the like are added to the N-terminus of the protein to form a fusion protein. The obtained recombinant expression vector is identified through DNA sequencing to confirm that the protein gene fragment is correctly contained. It should be noted that adding the tag is only for the convenience of identifying and extracting the protein, and does not have a decisive influence on the function of the protein as an antigen. When used, no tag is added or other tags are added or other labeling / identifying means are adopted.

[0023] The recombinant plasmid containing the protein gene fragment is transformed into the E. coli BL21 (DE3) competent cells, and the clones are inoculated into LB culture medium and cultured at 37°C on a shaker. When the bacterial density OD 600 is about 0.8, the temperature is lowered to 16°C, 0.1 mM isopropyl thiogalactoside (IPTG) is added to each LB culture medium, expression is induced overnight, and the bacterial body is obtained.

[0024] The bacteria bodies of the induced expression were collected by centrifugation and rinsed twice with PBS. The bacteria bodies were resuspended and dispersed with lysis solution (5-10 mL of lysis solution per g of bacteria bodies), ice-bathed, and ultrasonically broken (ultrasonic power 200 W, breaking for 5 s and resting for 5 s). After breaking, the bacteria bodies were centrifuged at 13000 rpm and 10°C for 20 min, and the supernatant was taken. After two-step purification of Ni column affinity chromatography and molecular sieve chromatography, the protein was confirmed for molecular weight and purity by SDS-PAGE electrophoresis analysis, and the concentration was determined by the Bradford method. After storage at -80°C, the purified candidate proteins were obtained. Among them, the full proteins of cTnI, TNNC1 and HSP65 were obtained; the protein fragments of HBEGF (19-160), NPPA (26-151), DCD (20-110) and NPY (29-97) were obtained; and the full protein of VEGFR1 was not obtained.

[0025] In this embodiment, the solutions and reagents used are as follows:

[0026] (2) Blocking solution / sample diluent / antibody diluent: 10 g of BSA (bovine serum albumin) was dissolved in 800 ml of coating buffer, and the coating buffer was added to 1 L.

[0027] (3) Washing solution: prepared immediately before use, 0.5% Tween 20 (V / V) was added to the coating buffer before use, pH = 7.4.

[0028] (4) TMB color developing agent, purchased from KPL company.

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

[0030] 2. Solid-phase coating of the protein to be tested. The purified candidate proteins obtained in Example 1 were diluted to 5 μg / mL with coating buffer, added to a 96-well plate at 50 μL per well, and coated at 4°C overnight. The next day, the solution was poured out, spun dry, and washed three times with washing solution at 200 μL per well each time. Then 200 μL of blocking solution was added to each well, incubated at room temperature for 1 h, the blocking solution was poured out, spun dry, and washed three times with washing solution at 200 μL per well each time and spun dry again; solid-phase coated antigens located in the 96-well plate were obtained.

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

[0032] 4. Add the enzyme-labeled secondary antibody. Dilute 1.0 mg / mL of horseradish peroxidase-labeled recombinant goat anti-human immunoglobulin G antibody (purchased from Jackson ImmunoResearch Inc.) 20,000 times with the antibody diluent, and then add 50 μL of the diluted antibody 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 hour with shaking. After that, spin dry, wash three times with 200 μL of washing solution per well, and spin dry again.

[0033] 5. Color development and reading of optical density values. Add 50 μL of TMB color developing agent to each well of the 96-well plate treated in step 4, shake for 15 seconds, and develop color at room temperature for 15 minutes in the dark. Then, add 50 μL of a stop solution, and read the absorbance values at 450 nm using an enzyme-labeled instrument to obtain the detection signal (S) of each sample to be tested.

[0034] 6. Sensitivity and specificity analysis. Take 384 positive samples (serum samples of patients who have been diagnosed as having myocardial infarction) and 384 negative samples (serum samples of healthy subjects) respectively, and determine the detection signal (S) of each sample according to the above method (absorbance values at 450 nm). Take the negative samples as negative reference samples, calculate the average (M) and standard deviation (SD) of the detection signals (S) of all the negative reference samples, and take M+3SD as the Cut Off value. Take the samples with a detection signal (S) ≥ the Cut Off value (S≥M+3SD) as positive, and take the samples with a detection signal (S) < the Cut Off value (S

[0035] Calculate the specificity and sensitivity based on the positive and negative results of the samples. The specificity refers to the proportion of healthy subject samples that are correctly determined to be negative, i.e., the number of samples determined to be negative among the negative samples divided by the total number of negative samples. The sensitivity refers to the proportion of positive patient samples that are determined to be positive, i.e., the number of samples determined to be positive among the positive samples divided by the total number of positive samples. Calculate the sensitivity and specificity when each protein to be tested is used as an antigen for sample testing. The results are shown in Table 2. " / " indicates that the corresponding protein is not obtained, and no determination is performed.

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

[0037] Results show that most of the potential proteins or protein fragments have low sensitivity in detecting acute myocardial infarction alone, and HSP65 has relatively higher sensitivity but low specificity, resulting in high false positive rate; and it is difficult to be applied clinically.

[0038] 7. Analyze the amino acid sequences and structures of the whole proteins of cTnI, HSP65 and VEGFR1. After a large number of preliminary tests, select different sequence fragments, and determine the sensitivity and specificity of detecting acute myocardial infarction according to the method of step 6. The selected sequence fragments and detection results are shown in Table 3.

[0039] Table 3 Comparison of protein fragments and detection results

[0040] Results show that compared with the whole protein and other protein fragments, the sensitivity of cTnI (31-164) is significantly improved while maintaining high specificity. The sensitivity of HSP65 (187-375) is slightly lower than that of HSP65 whole protein and HSP65 (1-540), but the specificity is significantly improved, which can reduce the possibility of false positives while ensuring the detection rate, so HSP65 (187-375) is selected as the preferred fragment. The specificity of VEGFR1 (781-1338) and VEGFR1 (27-242) is comparable, and the sensitivity detection values are not significantly different, but the AUC area of VEGFR1 (781-1338) is larger, which can reduce the possibility of false positives, so VEGFR1 (781-1338) is selected as the preferred fragment.

[0041] 8. Use the protein fragments in Table 3 to detect 96 cases of confirmed coronary heart disease patients and 96 cases of healthy controls with the same age and gender as the coronary heart disease cases using indirect enzyme-linked immunoassay (detection method steps 1-6, only the samples are replaced with the samples in this step). The coronary heart disease patients include 50 males and 46 females, aged 40-79 years, with an average age of 54 years. The diagnosis method of the patients conforms to the clinical diagnosis standard of coronary heart disease of the World Health Organization (WHO). The samples of the coronary heart disease patients were collected immediately after the diagnosis.

[0042] Results show that compared with the serum reaction of normal people, compared with the whole protein and other protein fragments, the antibodies produced by cTnI (31-164), HSP65 (187-375) and VEGFR1 (781-1338) can respectively induce more obvious antigen-antibody reaction in serum (manifested as significant increase in OD value). It is proved that these protein fragments have the potential to screen and detect coronary heart disease.

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

[0044] Table 4: Candidate proteins (fragments) and detection result comparison table

[0045] Example 3: Effect of protein composition in combination with detection of acute myocardial infarction samples

[0046] The "double index" method refers to: when a sample to be detected is detected using an antigen combination, if the detection signals of two antigens in the antigen combination are both positive detection signals, the detection result of the sample is positive, otherwise it is negative. The rest of the operation and the definition of sensitivity and specificity are the same as in Example 2. The results are shown in Table 5.

[0047] Table 5: Detection result comparison table of double antigen combination

[0048] The results show that: when using the "double index" method to detect acute myocardial infarction with a double antigen combination, the specificity is very high, and the problem of false positives can be basically avoided, but the positive detection rate is low, and the sensitivity is not ideal. Among them, the sensitivity of combinations 2, 6, 9, 17, 22 and 25 is relatively higher, and these combinations are selected for further experiments.

[0049] 3, increase the protein in the antigen combination, according to the "double index" method of step 2 (in this step, if two or more antigen detection signals are positive, it is judged to be positive, otherwise it is negative), using the samples of Example 2 and the sensitivity and specificity determination method, to detect the specificity and sensitivity of the antigen combination. The results are shown in Table 6. It should be noted that the inventors obtained the antigen combinations shown in Table 6 after a large number of preliminary experiments, not just the combinations in Table 6. Due to space limitations, only some of the combinations with better results are selected here.

[0050] Table 6: Detection result comparison table of antigen combination for acute myocardial infarction

[0051] The results show that: combinations 1-13 can significantly improve the detection rate and sensitivity while maintaining high specificity, effectively improving the clinical application value.

[0052] The above examples only describe the preferred mode of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications, variations, modifications and replacements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An antigen combination, characterized by: The antigen combination at least comprises a cTnI (31-164) protein fragment, and an amino acid sequence of the cTnI (31-164) protein fragment is shown as SEQ ID NO:

1.

2. The antigen combination according to claim 1, characterized in that: The antigen combination further comprises any one or both of a DCD (20-110) protein fragment and a TNNC1 whole protein, an amino acid sequence of the DCD (20-110) protein fragment is shown as SEQ ID NO: 6, and an amino acid sequence of the TNNC1 whole protein is shown as SEQ ID NO:

3.

3. The antigenic combination according to claim 2, characterized in that: The antigen combination comprises both the DCD (20-110) protein fragment and the TNNC1 whole protein, and further comprises: an HBEGF (19-160) protein fragment or an NPY (29-97) protein fragment, an amino acid sequence of the HBEGF (19-160) protein fragment is shown as SEQ ID NO: 2, and an amino acid sequence of the NPY (29-97) protein fragment is shown as SEQ ID NO:

8.

4. The antigenic combination according to claim 2, characterized in that: The antigen combination comprises both the DCD (20-110) protein fragment and the TNNC1 whole protein, The antigen combination further comprises both: an HSP65 (187-375) protein fragment and an HBEGF (19-160) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment and an HBEGF (19-160) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment, an HBEGF (19-160) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; An amino acid sequence of the HSP65 (187-375) protein fragment is shown as SEQ ID NO: 5, an amino acid sequence of the HBEGF (19-160) protein fragment is shown as SEQ ID NO: 2, an amino acid sequence of the VEGFR1 (781-1338) protein fragment is shown as SEQ ID NO: 7, an amino acid sequence of the NPPA (26-151) protein fragment is shown as SEQ ID NO: 4, and an amino acid sequence of the NPY (29-97) protein fragment is shown as SEQ ID NO:

8.

5. The antigenic combination according to claim 2, characterized in that: The antigen combination comprises both the cTnI (31-164) protein fragment and the DCD (20-110) protein fragment, Further comprises both: an HBEGF (19-160) protein fragment and an NPY (29-97) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment and an HBEGF (19-160) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; Or further comprises both: an HSP65 (187-375) protein fragment, a VEGFR1 (781-1338) protein fragment, an HBEGF (19-160) protein fragment, an NPPA (26-151) protein fragment and an NPY (29-97) protein fragment; or; also simultaneously comprising: HSP65(187-375) protein fragment, HBEGF(19-160) protein fragment and NPY(29-97) protein fragment; or; also simultaneously comprising: HBEGF(19-160) protein fragment, NPPA(26-151) protein fragment and NPY(29-97) protein fragment; the amino acid sequence of the HBEGF(19-160) protein fragment is shown as SEQ ID NO: 2, the amino acid sequence of the NPY(29-97) protein fragment is shown as SEQ ID NO: 8, the amino acid sequence of the HSP65(187-375) protein fragment is shown as SEQ ID NO: 5, and the amino acid sequence of the NPPA(26-151) protein fragment is shown as SEQ ID NO:

4.

6. The antigenic combination according to claim 1, characterized in that: the antigen combination comprises cTnI(31-164) protein fragment, HSP65(187-375) protein fragment, TNNC1 full protein and NPY(29-97) protein fragment; or; the antigen combination comprises cTnI(31-164) protein fragment, HSP65(187-375) protein fragment, VEGFR1(781-1338) protein fragment, TNNC1 full protein, NPPA(26-151) protein fragment and NPY(29-97) protein fragment; the amino acid sequence of the HSP65(187-375) protein fragment is shown as SEQ ID NO: 5, the amino acid sequence of the TNNC1 full protein is shown as SEQ ID NO: 3, the amino acid sequence of the NPY(29-97) protein fragment is shown as SEQ ID NO: 8, the amino acid sequence of the VEGFR1(781-1338) protein fragment is shown as SEQ ID NO: 7, and the amino acid sequence of the NPPA(26-151) protein fragment is shown as SEQ ID NO:

4.

7. Use of the antigen combination of any one of claims 1-6 in the preparation of a product for detecting / identifying acute myocardial infarction.

8. A reagent or kit for detecting / identifying acute myocardial infarction prepared by using the antigen combination of any one of claims 1-6.

9. A reagent or kit for detecting / identifying acute myocardial infarction comprising the antigen combination of any one of claims 1-6.

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