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A method for functionally detecting antibody-mediated organ rejection and its application and kit
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A technology for detecting antibodies and functionalities, applied in the biological field, can solve the problems that organs cannot perform their expected functions, increase the economic burden of patients, etc., and achieve the effect of high sensitivity and accuracy
Active Publication Date: 2018-12-11
石强
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[0007] To sum up, chronic antibody-mediated rejection is the primary problem to be solved in organ transplantation medicine today. It not only increases the financial burden of patients, but also makes the transplanted organs unable to perform the expected functions in the body
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Embodiment 1
[0072] Example 1 Separation of vascular endothelial cells
[0073] Solid tissues obtained aseptically through surgical procedures are placed in rich media and transferred to the processing room within 3-5 hours. Depending on the nature of the entity organization, different solutions are adopted.
[0074] (1) For incomplete tissues such as blood vessel fragments, fat, muscle, skin, some organs, and umbilical cord, cells will be collected by direct digestion. The specific steps are: cut the tissue into pieces of 1 cubic millimeter, wash and remove all the blood, place the tissue in a culture medium containing 1% collagenase by volume, and incubate at 37°C for 0.5-1 hour , mechanically loosen the digested tissue and separate single cells and residual tissue with gauze. Collagenase was removed using centrifugation.
[0075] (2) For intact organs with vascular connections such as liver, lung, kidney, etc., collect vascular endothelial cells by organ perfusion. According to the ...
Embodiment 2
[0076] Example 2 Culture of vascular endothelial cells
[0077] The single cell suspension obtained in Example 1 was placed in a mixture containing 20% fetal bovine serum by volume, 15 mg / ml endothelial cell growth stimulating supplement (ECGS), 50 units / ml heparin, 100 units / ml penicillin-100 units / ml M1 streptomycin culture medium, inoculated in a culture bottle coated with 1% gelatin, placed at 35°C and 4% CO by volume. 2 Culture in a saturated humidity incubator, and replace the culture medium after 24 hours.
Embodiment 3
[0078] Example 3 Cryopreservation of vascular endothelial cells
[0079] When the growth of the cells reached to cover 90% of the surface area of the culture flask, the cells were detached from the surface of the culture flask with a concentration of 0.05% trypsin-ethylenediaminetetraacetic acid disodium salt (EDTA) digestion solution. After centrifuging to remove residual enzymes, suspend the cells in a culture medium containing 5% DMSO by volume, gradually reduce the temperature of the above liquid to -80°C at a rate of 1-2°C / min, and then transfer to liquid nitrogen Store in can.
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Abstract
The invention discloses a method for functionally detecting antibody-mediated organ rejection, its application and a kit, which relate to the field of biotechnology. The invention is used to determine whether antibodies leading to chronic organ rejection are produced in patients receiving organ transplantation, and the kit is used to detect pathogenic specific anti-donor antibodies. In the present invention, the serum of the organ recipient is added into the same vascular endothelial cell culture as the donor's histocompatibility antigen, and the reaction of the vascular endothelial cells is measured after culturing at a temperature of 35°C-37°C. The results of the experiment revealed a highly specific and precise interaction between organ transplant recipients and donors, providing a pathological basis for early detection of organ rejection and formulation of early treatment plans. Without the need for biopsy materials, it provides clinicians with a powerful, effective and economical means of regularly monitoring organ transplant patients.
Description
technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for functionally detecting antibody-mediated organ rejection and its application and kit, which are used to determine whether there is a chronic organ rejection in a patient receiving organ transplantation. Antibody. Background technique [0002] Surgical transplantation is currently the most effective way to cure organ failure. It not only requires strict and extensive medical care, but also requires the same human histocompatibility antigen between the organ donor and recipient to prevent the recipient's immune system from attacking foreign tissues. If the two types of human histocompatibility antigens are different, the recipient's naturally occurring or induced synthetic antibodies will attack the transplanted organ tissue. However, in clinical practice, organ donors and resources are very limited, and the degree of antigenicity between donors and recipients var...
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