Antibody for detecting early damage of blood-brain barrier in ischemic stroke and its application
A technology for ischemic stroke and blood-brain barrier, applied in the field of disease detection, can solve problems such as misjudgment, inability to rule out the influence of occludin full-length protein, and affect the accuracy of test results, so as to eliminate the influence, improve specificity and accuracy rate effect
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Embodiment 1
[0054] The development of embodiment 1 specific antibody
[0055] This example is used to illustrate the development process of the specific antibody of the present invention, and the specific steps are as follows:
[0056] 1. According to the human occludin full-length protein sequence (AAB00195.1, as shown in SEQ ID NO.1), after hydrophobicity analysis ( figure 1 ), judge the protein as multiple transmembrane protein, and predict the suitable polypeptide sequence for antigen recognition and antibody preparation. From the N-terminal to the C-terminal, select 4 sequences for peptide synthesis.
[0057] Sequence 1#YRPDEFKPNHYAPSNC;
[0058] Sequence 2#KTRRKMDRYDKSNIL;
[0059] Sequence 3#DHYETDYTTGGESC;
[0060] Sequence 4#SKLSHIKKMVGDYDR.
[0061] 2. Synthetic antigen:
[0062] Coupling the above-mentioned 1#~4# polypeptides with KLH respectively (the coupling agent is SuLfo-SMCC) to obtain KLH-polypeptides as 1#~4# immune antigens;
[0063] The above-mentioned 1#-4# po...
Embodiment 2
[0076] Example 2 Kit for specific detection of early damage to the blood-brain barrier in ischemic stroke
[0077] This example takes the specific antibody XW-OCLN-2 obtained in step 4 of Example 1 as an example to illustrate a kit for specifically detecting early damage to the blood-brain barrier in ischemic stroke. The kit is an ELISA kit, including : Solid phase carrier coated with specific antibody XW-OCLN-2, detection antibody, enzyme-labeled antibody, standard antigen.
[0078] in:
[0079] The preparation method of the solid phase carrier is specifically:
[0080] (1) Coating: with 200mM NaHCO 3 Dilute the specific antibody XW-OCLN-2 in the buffer solution to a final concentration of 1 μg / mL, add 100 μL occludin specific antibody diluent to each well of the microtiter plate, and refrigerate overnight at 4 °C;
[0081] (2) Washing: wash each well of the microplate with PBST 4 times, 5 min each time;
[0082] (3) Blocking: add blocking solution 10% BSA, incubate at 37...
Embodiment 3
[0088] The application of embodiment 3 detection kits
[0089] This example is used to illustrate how to use the ELISA kit prepared in Example 2 to detect the level of occludin-specific degradation fragments in the serum of patients with acute ischemic stroke, so as to measure the early damage of the blood-brain barrier in patients with stroke.
[0090] In this example, the ELISA kit prepared in Example 2 was used to detect the sera of 7 acute ischemic stroke patients and 6 healthy persons.
[0091] (1) Collect serum from patients with acute ischemic stroke and healthy patients; add 100 μL of human serum, incubate at 37°C for 2 hours, and wash;
[0092] (2) Add 100 μL detection antibody (Thermo Fisher Cat. No. 33-1500), incubate for 2 hours, and wash 4 times;
[0093] (3) Add 100 μL of secondary antibody (Zhongshan Jinqiao ZB-2305), incubate at 37°C for 30 minutes, and rinse;
[0094] (4) Add 90 μL of TMB substrate chromogenic solution to each well, and develop color at 37°C...
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