Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof
A technology of cardiac troponin and hybridoma cells, which is applied in the field of immunoassay of cardiac troponin I, can solve the problems of inconvenient use, large investment, narrow linear range, etc.
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Embodiment 1
[0027] Embodiment 1 The establishment of hybridoma cells and the preparation of anti-cardiac troponin I monoclonal antibody
[0028] 1. Antigen Immunization
[0029] Human recombinant cardiac troponin I antigen (1.5mg / ml, produced by Shenzhen Faipeng Biological Co., Ltd., product number AG-CTNI-HP0005) and an equal volume of Freund's complete adjuvant (appearance: amber cell suspension; Components: Paraffin Oil85%, Mannide Monooleate15%, Mycobacterium smegmatis1mg / ml) are mixed to obtain an oily emulsion. The emulsion was subcutaneously administered to the back site of BALB / c mice (Guangzhou Provincial Experimental Animal Center, 6-week-old female, 5) at a dose of 0.2ml, and the abdominal cavity was boosted immunization 14 days after the first immunization (antigen and equal amount of Fu Mixed with incomplete adjuvant), after boosting immunization to four injections, the tail blood was collected for titer detection, and the titer reached the fusion requirement.
[0030] Thre...
Embodiment 2
[0067] The preparation of embodiment 2 cardiac troponin I colloidal gold detection test paper
[0068] 1. Preparation of Nitrocellulose Membrane
[0069] Preparation of coating buffer: 6% methanol, 0.01M pH7.2 PBS buffer as coating buffer, filtered through 0.22μm membrane, set at 4°C for later use, valid for one week. 1000ml 6% Methanol 0.01M pH7.2PBS Buffer Formula: NaCL8g, KCL0.2g, NaCL 2 HPO 4 12H 2 O2.9g, KH 2 PO 4 0.2g, methanol 60ml, distilled deionized water to 1000ml.
[0070] Preparation of nitrocellulose membrane: Dilute the anti-cardiac troponin I rabbit polyclonal antibody (manufactured by Shenzhen Faipeng Biological Co., Ltd., product number PAB-CTNI-AP0002) to 1-5 mg / ml with coating buffer, adjust Machine, marked as T line, which is the detection line, the T line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad; the goat anti-mouse IgG antibody (produced by Shenzhen Feipeng Biological Co., Ltd. , Cat. No. B...
Embodiment 3
[0094] Embodiment 3 detects the kit of cardiac troponin I
[0095] 1. The kit for rapid detection of cardiac troponin I comprises: the detection test paper and sample diluent made in Example 2.
[0096] Among them, the sample diluent is 8% NaCl solution. Preparation method: 80gNaCl, add distilled water to make up to 1000ml.
[0097] 2. Detection of cardiac troponin I by colloidal gold method
[0098] (1) Pipette 120 μl of collected human serum or plasma directly into the sample hole of the test paper card, and wait for 15 minutes to observe the results.
[0099] (2) Judgment of results: When the test strip shows a red quality control line visible to the naked eye, but no red detection line visible to the naked eye, the result is judged as negative; when the test strip shows a red quality control line visible to the naked eye, it also appears Visible red test line, the result is judged as positive. The darker the detection line, the higher the cardiac troponin I level of th...
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