Detection method for cat trace lymphopoiesis

Abstract

The invention provides a cat trace lymphopoiesis detection method which comprises the following steps: uniformly mixing receptor cat serum and donor cat lymphocytes for first incubation, adding complement after the first incubation is completed, uniformly mixing for second incubation, and obtaining a to-be-detected group after the second incubation is completed; uniformly mixing a phosphate buffer solution and donor cat lymphocytes for first incubation, adding complement after the first incubation is completed, uniformly mixing for second incubation, and obtaining a blank control group after the second incubation is completed; and after the second incubation is completed, respectively adding a coloring agent into the group to be detected and the blank control group, uniformly mixing, respectively counting the death rate of donor cat lymphocytes of the group to be detected and the death rate of donor cat lymphocytes of the blank control group, and calculating the toxicity percentage of the cat lymphocytes by adopting a specific formula. By setting a blank control group, non-specific death interference is eliminated by adopting a cat lymphocyte poison calculation formula, so that the credibility of a detection result is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a method for detecting cat micro-lymphocyte toxicity. Background Art

[0002] With the continuous development of modern medical technology, organ transplantation has become one of the most important means of treating terminal diseases of various organs and tissues in the body. Currently, cat organ transplantation in China is in its early stages of development and faces many challenges. Among them, the recipient's innate immune system can react to allogeneic organs and tissues in various ways. Among them, immune rejection reactions involving feline leukocyte antigen (FLA) and lymphocytes (Lym) are one of the most important factors affecting the success of organ transplantation.

[0003] Immune rejection is the process by which the donor graft is recognized and attacked by the recipient's immune system after transplantation. If the recipient has received blood transfusions, immunotherapy, or other treatments prior to surgery, pre-existing antibodies against the donor's Major Histocompatibility Complex (MHC) may be present, leading to rejection. The key processes involved include "foreign self recognition," "immune activation," and "foreign self elimination." Therefore, allogeneic transplantation carries the risk of rejection. To mitigate this risk, thorough pre-transplant assessment is essential. Complement-dependent cytotoxicity (CDC) testing is considered one of the gold standards in human transplantation. This method uses an in vitro simulation of the rejection reaction to assess the donor-recipient match before transplantation. This method determines whether the recipient is compatible with the donor graft, thereby minimizing post-transplant rejection and prolonging graft survival. CDC, also known as complement-dependent cytotoxicity testing, is a mandatory pre-transplant assessment in clinical organ transplantation. The principle is that complement binds to the Fc segment of antibodies in the recipient cat's serum, activates complement activity, and forms a membrane attack complex. This substance can form channels on the donor cat's lymphocyte membrane, allowing only water and electrolytes to pass through, ultimately leading to changes in intracellular osmotic pressure, cell membrane rupture, and a cell-killing effect.

[0004] China's cat organ transplant program is in its early stages of development. To improve the safety and success rate of transplantation procedures, there is an urgent need to establish a standardized feline lymphocytotoxicity test (CDC) system for preoperative donor screening and immune risk assessment. Due to its ease of use and minimal equipment requirements, this test has strong potential for clinical adoption and is expected to become a key testing tool in pet healthcare. Summary of the Invention

[0005] The object of the present invention is to provide a method for detecting cat micro-lymphocyte toxicity in response to the gaps in the prior art, comprising:

[0006] The recipient cat serum and the donor cat lymphocytes are mixed and incubated for the first time. After the first incubation is completed, complement is added and mixed and incubated for the second time. After the second incubation is completed, the test group is obtained;

[0007] Phosphate buffer and donor cat lymphocytes were mixed and incubated for the first time. After the first incubation, complement was added and mixed and incubated for the second time. After the second incubation, a blank control group was obtained.

[0008] After the second incubation is completed, dye is added to the test group and the blank control group and mixed, and the mortality rate of cat lymphocytes of the donors in the test group and the blank control group are counted respectively. The percentage of cat lymphocyte cytotoxicity is obtained based on the mortality rate of cat lymphocytes of the donors in the test group and the blank control group.

[0009] Furthermore, the specific method for obtaining the lymphocyte cytotoxicity percentage based on the lymphocyte mortality rate of the cat donors in the test group and the blank control group is as follows:

[0010] Feline Lymphocytotoxicity (%Cytotoxicity) = [(% Dead Lymphocytes in Test Group - % Dead Lymphocytes in Blank Control Group) / (100% - % Dead Lymphocytes in Blank Control Group)] × 100%; where the % Dead Lymphocytes in Test Group represents the mortality of donor feline lymphocytes in response to recipient feline serum, and the % Dead Lymphocytes in Blank Control Group represents the mortality of donor feline lymphocytes in response to phosphate-buffered saline (PBS). Phosphate-buffered saline (PBS) does not contain antibodies and cannot activate complement. The % Dead Lymphocytes in the Blank Control Group reflects spontaneous complement activation or nonspecific damage, such as mechanical injury from handling. Therefore, this formula excludes nonspecific death (such as spontaneous complement activation or handling damage) and retains only the antibody-dependent complement killing effect.

[0011] Furthermore, the content of the donor cat lymphocytes was adjusted to 2×10 6 cells / L. Standardized cell density avoids insufficient complement consumption due to excessively high concentrations or weakened signals due to excessively low concentrations, ensuring a dynamic balance of the antibody-complement-cell reaction and improving data comparability.

[0012] Furthermore, the volume ratio of the recipient cat serum to the donor cat lymphocytes is 1:1. The antigen-antibody binding ratio is optimized to avoid excessive serum inhibition of complement activity or excessive cell dilution of antibody concentration, thereby ensuring the stoichiometric balance of the reaction system.

[0013] Furthermore, the volume ratio of complement, serum and donor lymphocytes is 1:1:1. Complement and antibodies form an optimal synergistic ratio to avoid excessive complement causing nonspecific lysis (such as direct cell lysis by complement) or insufficient complement causing insufficient killing efficacy.

[0014] Furthermore, the complement is rabbit complement or guinea pig complement. Rabbit complement is highly effective in activating the cat antibody system, while guinea pig complement has wider compatibility.

[0015] Furthermore, the first incubation and the second incubation are completed in a 37°C water bath for 10-20 minutes. This simulates physiological temperature to accelerate antigen-antibody binding and complement cascade reactions, and precisely controls the time to prevent excessive incubation leading to natural cell apoptosis, thereby balancing reaction efficiency and specificity.

[0016] Furthermore, the cell viability of the donor cat lymphocytes was adjusted to >90% before the test. High cell viability reduces experimental background noise (such as spontaneous absorption of trypan blue by dead cells), ensuring that the observed death is mainly mediated by antibodies and complement rather than sample quality issues.

[0017] Furthermore, the stain is a trypan blue staining solution. Trypan blue only penetrates the membranes of dead cells, allowing for rapid differentiation of live and dead cells. Compared with fluorescent staining, it is less expensive and simpler to perform, making it suitable for use in primary healthcare institutions.

[0018] Furthermore, when calculating lymphocyte mortality in the cats in the test group and the blank control group, complete the lymphocyte count within 5 minutes to record the mortality rate. Trypan blue is cytotoxic, and prolonged staining can lead to cell death. Rapid counting avoids morphological changes that interfere with interpretation, ensuring the timeliness and accuracy of mortality statistics.

[0019] The beneficial effects of the present invention are:

[0020] 1. By setting up a blank control group and using the cat lymphocyte cytotoxicity calculation formula to eliminate non-specific death interference (such as spontaneous complement activation or operational damage), the antibody-dependent cytotoxicity can be accurately quantified, significantly improving the credibility of the test results.

[0021] 2. By controlling the donor lymphocyte viability to >90%, optimizing the complement concentration and reaction ratio, we can minimize background interference, improve the sensitivity of antibody recognition, and ensure the detection rate of weakly positive samples.

[0022] 3. Allows the selection of rabbit or guinea pig complement to adapt to different laboratory resource conditions; the micro-sample requirement (microliter level) reduces experimental costs and facilitates promotion in the field of pet medicine. DETAILED DESCRIPTION

[0023] The raw materials used in the following examples are all commercially available products unless otherwise specified, and the methods used are all conventional methods in the art unless otherwise specified. The present invention is further illustrated by the following examples.

[0024] Preparation of complement, recipient cat serum, and donor cat lymphocytes: Collect blood from 10 or more rabbits or guinea pigs. Place the blood in a 37°C water bath for 30 minutes to separate the serum. Collect the serum with a small amount of red blood cells and centrifuge it at 3000 rpm. Mix the supernatants to form a mixed complement and store it at 4°C or -80°C. Collect serum from the recipient cat to be tested and whole blood from the donor cat to be tested. Use the Ficoll method to separate the donor lymphocytes to be tested and adjust the lymphocyte count to 2×10 6 cells / L, ensuring cell viability >90%.

[0025] Preparation of the test group: Add 100 μL of donor cat lymphocytes to a blank tube, add 100 μL of the test serum, pipette with a 200 μL pipette tip, mix thoroughly, and incubate in a 37°C water bath for 10 minutes. Add fresh rabbit mixed complement or guinea pig mixed complement (100 μL) to the mixture after the first incubation, pipette and mix thoroughly, and incubate in a 37°C water bath for a second time for 20 minutes. Mix 0.4% trypan blue staining solution with the mixture after the second incubation at a ratio of 1:9 (trypan blue staining solution concentration is 0.04%). Complete cell counting within 5 minutes and record cell mortality.

[0026] Preparation of the blank control group: Take donor cat lymphocytes (100 μL) and add them to a blank tube. After adding phosphate buffer (100 μL), pipette with a 200 μL pipette tip, mix thoroughly, and place in a 37°C water bath for the first incubation for 10 minutes. Add fresh rabbit mixed complement or guinea pig mixed complement (100 μL) to the mixture after the first incubation, pipette thoroughly to mix, and place in a 37°C water bath for a second incubation for 20 minutes. Mix 0.4% trypan blue staining solution with the mixture after the second incubation at a ratio of 1:9 (final concentration of trypan blue staining solution is 0.04%). Complete cell counting within 5 minutes and record cell mortality.

[0027] Finally, the lymphocyte cytotoxicity percentage was calculated according to the formula: CP% = [(% of dead lymphocytes in the test group -% of dead lymphocytes in the blank control group) / (100% -% of dead lymphocytes in the blank control group)] × 100%. The final CP% was the lymphocyte cytotoxicity percentage.

[0028] The percentage of lymphocytotoxicity is graded as follows:

[0029] Lymphocytotoxicity percentage Grading ＜10％ - 11％～20％ ± 21％～30％ + 31％～50％ ++ 51％～80％ +++ ＞80％ ++++

[0030] Transplant surgery generally requires that the number of dead cells is less than 10%, and the smaller the number, the better. If it is higher than 20%, hyperacute rejection reaction may occur after transplantation.

[0031] The above-described embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present application, and should all be included in the scope of protection of the present application.

Claims

1. A method for detecting cat micro-lymphocyte toxicity, characterized in that: include: The recipient cat serum and the donor cat lymphocytes are mixed and incubated for the first time. After the first incubation is completed, complement is added and mixed and incubated for the second time. After the second incubation is completed, the test group is obtained; Phosphate buffer and donor cat lymphocytes were mixed and incubated for the first time. After the first incubation, complement was added and mixed and incubated for the second time. After the second incubation, a blank control group was obtained. After the second incubation is completed, dye is added to the test group and the blank control group and mixed, and the mortality rate of cat lymphocytes of the donors in the test group and the blank control group are counted respectively. The percentage of cat lymphocyte cytotoxicity is obtained based on the mortality rate of cat lymphocytes of the donors in the test group and the blank control group.

2. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The specific method for obtaining the percentage of lymphocyte cytotoxicity based on the lymphocyte mortality rate of the donor cats in the test group and the blank control group is as follows: Feline lymphocyte cytotoxicity % = [(% of dead lymphocytes in the test group - % of dead lymphocytes in the blank control group) / (100% - % of dead lymphocytes in the blank control group)] × 100%; Among them, the dead lymphocyte rate (%) of the test group is the death rate of donor cat lymphocytes under the action of recipient cat serum, and the dead lymphocyte rate (%) of the blank control group is the death rate of donor cat lymphocytes under the action of phosphate buffer.

3. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein Before the test, the content of the donor cat lymphocytes was adjusted to 2×10 6 cells / L.

4. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The volume ratio of the recipient cat serum to the donor cat lymphocytes is 1:

1.

5. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The volume ratio of the complement, serum and donor lymphocytes is 1:1:

1.

6. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The complement is rabbit complement or guinea pig complement.

7. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The first incubation and the second incubation were completed by incubating in a 37° C. water bath for 10-20 minutes.

8. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The cell viability of the donor cat lymphocytes was adjusted to >90% before the test.

9. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein The staining agent is trypan blue staining solution.

10. The method for detecting cat micro-lymphocyte toxicity according to claim 1, wherein: When counting the lymphocyte mortality rate of the donor cats in the test group and the blank control group, the lymphocyte count should be completed within 5 minutes to record the lymphocyte mortality rate.