Enzyme linked immune detection method of patient thrombocyte antibody and cross matching type
An enzyme-linked immunoassay and enzyme-linked immunosorbent technology, which is applied in the medical field, can solve problems such as increased false positive probability, limited antigen detection, narrow coverage, etc., and achieves improved safety and effectiveness, improved sensitivity, and high practicality. The effect of applying value
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Embodiment 1
[0020] Embodiment 1, platelet membrane antigen complex (OMPC) immunization rabbit obtains the purification of polyclonal antiserum and polyclonal antibody
[0021] 1. Preparation of platelet suspension, extraction and identification of outer membrane protein complex
[0022] 1. Preparation of platelet suspension
[0023] Take 50mL of human platelet-rich plasma (PRP) in a centrifuge tube, centrifuge at 3000rpm at 22°C for 20min, and wash the precipitate with TEGS (Tris 20mmol / L, EDTA 0.6mmol / L, Glu 5mmol / L, NaCl 148mmol / L, pH7.4 ) fully washed 3-5 times to remove red blood cells and white blood cells as much as possible, and finally adjust the platelet concentration to 5×10 with normal saline 9 per mL, and stored at -20°C after aliquoting.
[0024] 2. Extraction of Platelet Outer Membrane Protein Complex (OMPC)
[0025] The following four methods were used to extract the platelet outer membrane protein complex (OMPC):
[0026] 1) SDS crushing method: adjust the concentratio...
Embodiment 2
[0071] Embodiment 2, the establishment and condition optimization of the ELISA method of detection patient's platelet antibody and cross-match type
[0072] 1. Determination of the optimum platelet suspension concentration
[0073] The optimum concentration of the platelet suspension is determined by titration by square array method, and the specific method includes the following steps:
[0074] 1) Platelets were serially diluted with coating solution (1×10 8 -5×10 8 platelets / mL), add 100ul platelet suspension to each hole for coating, block with 5% BSA, and wash 3 times;
[0075] 2) Select a strong positive (20ug / mL) and a weak positive (2ug / mL) polyclonal antibody obtained by immunizing rabbits with the purified platelet membrane antigen complex in Example 1 and rabbit serum before immunization as negative controls, and use Antibody diluents were diluted 1:100, added, incubated (room temperature 2h), washed 3 times;
[0076] 3) Add goat anti-rabbit enzyme-labeled second...
Embodiment 3
[0095] Embodiment 3, ELISA indirect method is used to detect the specificity verification experiment of BPC antibody
[0096] 1. In order to verify the specificity of the ELISA indirect method for detecting BPC antibodies, a specific absorption test is now carried out. The specific method is: select 500ul of 8 parts of ITP serum, add the same amount of BPC suspension (the isolated platelet suspension is adjusted to 1×10 with TEGS) 9 / L), mix well and place at 37°C for half an hour, then place at 4°C for 2-24h, centrifuge after absorption and take the supernatant according to the above experimental conditions and methods for ELISA determination, the results are shown in Table 3:
[0097] OD value of 8 positive sera
1
2
3
4
5
6
7
8
not absorbed
after absorption
inhibitory junction
fruit
1.588
1.366
+
1.230
0.980
+
1.220
0.917
+
1.555
1...
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