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Fluorescence in-situ micro-lymphocytotoxicity detection method and kit

A technology of micro-lymphatic toxicity and detection method, which is applied in the field of immunology, can solve the problems of poor sensitivity, false negative reaction, error, etc., and achieve the effects of improving accuracy and sensitivity, improving accuracy, and increasing the probability of complement binding

Inactive Publication Date: 2014-07-23
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this traditional CDC method is prone to false negative reactions or suspected negative reactions when the titer of the recipient's serum is low, resulting in large errors in the test results and poor sensitivity
At the same time, the traditional CDC method uses trypan blue staining to count the death rate of lymphocytes. This single staining method is easy to cause large errors due to human experience, which further reduces the accuracy of the CDC method.

Method used

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  • Fluorescence in-situ micro-lymphocytotoxicity detection method and kit
  • Fluorescence in-situ micro-lymphocytotoxicity detection method and kit
  • Fluorescence in-situ micro-lymphocytotoxicity detection method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: detection method of the present invention

[0023] The serum to be tested was routinely collected from the recipient, and the lymphocytes to be tested from the donor were separated by the Ficoll method, and the lymphocyte content was adjusted to 2×10 6 / L to ensure cell viability >90%.

[0024] Serum to be tested (2ul) + lymphocytes to be tested (1ul) were added to the reaction wells of the Terasaki plate, mixed slightly and then incubated at 20-25°C for 30min, so that the Fab fragment of the IgG antibody in the recipient serum and the surface antigen of the donor lymphocytes Fully combine, then add 8μl 1×PBS to each well, centrifuge at 1000rpm for 10sec, shake slightly, shake the plate, wash the plate twice, and wash away unbound substances.

[0025] Take out the aliquoted complement and mouse anti-human IgG κ light chain IgG (commercially available) from the -80°C refrigerator, thaw, and prepare immediately before use at a volume ratio of 1:150 (seconda...

Embodiment 2

[0027] Embodiment 2: detection method of the present invention

[0028] The serum to be tested was routinely collected from the recipient, and the lymphocytes to be tested from the donor were separated by the Ficoll method, and the lymphocyte content was adjusted to 2×10 6 / L to ensure cell viability >90%.

[0029] Serum to be tested (2ul) + lymphocytes to be tested (1ul) were added to the reaction wells of the Terasaki plate, mixed slightly and then incubated at 20-25°C for 30min, so that the Fab fragment of the IgG antibody in the recipient serum and the surface antigen of the donor lymphocytes Fully combine, then add 8μl 1×PBS to each well, centrifuge at 1000rpm for 10sec, shake slightly, shake the plate, wash the plate twice, and wash away unbound substances.

[0030] Take out the aliquoted complement and goat anti-human IgG κ light chain IgG (both purchased from One Lambda) from the -80°C refrigerator. preparation. Immediately add 5 ul of the freshly prepared complemen...

Embodiment 3

[0032] Embodiment 3: detection method of the present invention

[0033] The serum to be tested was routinely collected from the recipient, and the lymphocytes to be tested from the donor were separated by the Ficoll method, and the lymphocyte content was adjusted to 2×10 6 / L to ensure cell viability >90%.

[0034] Serum to be tested (2ul) + lymphocytes to be tested (1ul) were added to the reaction wells of the Terasaki plate, mixed slightly and then incubated at 20-25°C for 30min, so that the Fab fragment of the IgG antibody in the recipient serum and the surface antigen of the donor lymphocytes Fully combine, then add 8μl 1×PBS to each well, centrifuge at 1000rpm for 10sec, shake slightly, shake the plate, wash the plate twice, and wash away unbound substances.

[0035] Take out the aliquoted complement and rabbit anti-human IgG κ light chain IgG (commercially available) from the -80°C refrigerator, thaw, and prepare immediately before use at a volume ratio of 1:250 (second...

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Abstract

The invention relates to the field of immunology, and discloses a fluorescence in-situ micro-lymphocytotoxicity detection method and a kit. The method comprises the following steps of: combining a Fab fragment of IgG antibody in recipient serum with antigen on the surface of donor lymphocyte; washing a plate and then adding mixed liquid consisting of complement and anti-human IgG light chain secondary antibody for incubation, thereby combining the Fab fragment of the secondary antibody with the IgG antibody in the recipient serum; combining the complement with an Fc fragment of the secondary antibody; mediating a cell killing effect by the complement to kill off target cells; and performing fluorescent staining and counting the mortality of the donor lymphocytes by using a counting method. According to the detection method, the anti-human IgG light chain secondary antibody is increased and is used as a bridge to improve the complement fixation probability, the defect of difficulty in combining the complement with the IgG antibody caused by low titer of the recipient serum antibody existing in the conventional CDC (Micro-complement dependent cytotoxicity test) method is overcome, and the accuracy and the sensitivity for detecting the lymphocytotoxicity are improved; and the detection method can be applied to mating type detection before organ transplantation.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a fluorescence in-situ trace lymphotoxicity detection method and a kit. Background technique [0002] Organ transplantation has become one of the important means of treating organ failure, but this technology is also facing various challenges during the rapid development, in which the recipient's own immune system can recognize foreign organs, and human leukocyte antigen (Human Leukocyte Antigen) Leukocyte Antigen, HLA) is the main cause of immune rejection in organ transplantation. [0003] As a living being, the recipient has a natural ability and mechanism - the immune system, which can identify, control, destroy and eliminate the foreign "non-self" tissues and organs that enter the body. This physiological immune process is clinically manifested as immune rejection, leading to the destruction of transplanted organs and graft failure. Transplanted organs, just like other human cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/96
Inventor 李杨何军徐超
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV