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

Inactive Publication Date: 2006-06-28
马印图 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Coated platelets will dissolve, and the conformational epitope of the membrane antigen will change after dissolution, resulting in a decrease in the detection rate of autoantibodies and an increase in the probability of false positives
Even with MAIPA, the whole operation takes 4 hours, and the sensitivity and specificity are only 47% and 85%, and it requires monoclonal antibodies specific to platelet surface proteins. In some cases, the coverage of monoclonal antibodies is narrow, so Antigen detection is limited

Method used

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  • Enzyme linked immune detection method of patient thrombocyte antibody and cross matching type
  • Enzyme linked immune detection method of patient thrombocyte antibody and cross matching type

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

An enzyme ¿C linked immune detection method of patient blood platelet and cross ¿C match includes obtaining multiple clone antibody from blood platelet film antigen composite immune abimal and using it to carry out encapsulation then carry out closing, washing plate, adding patient sample, washing plate, adding substrate for color developing and stopping reaction.

Description

technical field [0001] The invention relates to a detection method in the medical field, in particular to an enzyme-linked immunological detection method for patient's platelet antibody and cross-matching type. Background technique [0002] In clinical blood transfusion, with the continuous promotion of component blood transfusion, blood therapy has attracted more and more attention and has become a research hotspot. How to quickly, safely and effectively transfuse blood is often related to the life of patients and is also the future development trend. Platelet transfusion can reduce severe bleeding caused by thrombocytopenia, and has become an effective supportive therapy for patients with hematological diseases and tumors with radiotherapy and chemotherapy. And platelet transfusion ineffective (RPT), so platelet antibody detection is very important in disease diagnosis and curative effect judgment. How to improve the clinical efficacy of platelet transfusion or avoid inef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/78
Inventor 马印图王全立
Owner 马印图
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