Rabbit serum with complementary activity for the typing of the main human histocompatibility complex
Mr. Walter Góngora Amores, I Dr. Rolando Sánchez Artigas, I Mr. Zuleidis Torres Ponce, I Dr. Javier Corella Sánchez, I Mr. Dailin Cobos Valdés, I Mr. Antonio Miranda Cruz, I Mr. Thamara Dunia Ricardo II
Center for Immunology and Biopreparations. Holguin, Cuba.
II Territorial Laboratory of Histocompatibility of the Medical University of Holguín. Holguin, Cuba.
In the microlymphocytotoxicity test, rabbit serum as a source of complement plays an essential role in the classification of the main human histocompatibility complex, both for antigenic typing and in the crossing of donor-recipient lymphocytes before performing the tests. transplants. In this work, 3 experimental batches of said blood derivative were obtained with optimal performance, biological activity and stability indicators.
The exsanguination of the animals was carried out using the jugulation technique. The serum was separated by centrifugation under controlled temperature and ambient conditions. Subsequently, the product was subjected to sterilizing filtration and samples were taken for quality tests. The study showed that the product retains biological activity for at least 18 months and that the cytotoxicity values are within acceptable limits for use in pretransplant histocompatibility assays.
Keywords: rabbit serum, complement activity, HLA typing, serological technique, microlymphocytotoxicity, organ transplantation.
The microlymphocytotoxicity assay is one of the serological methods used to classify the major human histocompatibility complex. In this technique, rabbit serum as a source of complement plays an essential role in the determination of antigens and in the lymphocyte cross-compatibility assay, a necessary step before selecting the best donor-recipient pair to perform the transplantation of organ. Three experimental batches of normal rabbit serum were developed with excellent indicators of efficiency, biological activity, and stability.
Rabbit blood was obtained by the jugulation technique and the serum was separated by centrifugation in an environment and controlled temperature conditions. The sterilization process was carried out by filtration and the samples were taken for quality control tests. Stability studies showed that the product maintained biological activity for at least 18 months after processing and the cytotoxicity values were adequate for use in pretransplant histocompatibility assays.
Keywords: rabbit serum, complement activity, HLA typing, serological method, micro lymphocytotoxic organ transplantation.
The main histocompatibility complex is known in man as human leukocyte antigens (HLA). The genes that encode these membrane proteins are found on the short arm of chromosome 6 and constitute the most polymorphic genetic complex that exists. 1
HLA typing has been of vital importance in transplant medicine and in the evaluation of the HLA-disease association. 2-4 Among the mechanisms that explain this relationship are the direct or indirect recognition by the receptor of non-compatible HLA molecules from the donor with the consequent stimulation of their immune response and the role that certain HLA specificities may play in the presentation of peptides endogenous. as an etiology of autoimmune diseases. 5
Among the methods for the determination of HLA antigens, serological ones and those based on molecular biology techniques stand out. In the former case, the microlymphocytotoxicity assay has become the standard serological technique for HLA typing and was developed by Terasaki and Mc Clelland in 1964. 6
Its principle is based on putting isolated lymphocytes from the individual to be studied (donor) in contact with a panel of alloantibodies or monoclonal anti-HLA capable of recognizing certain antigenic specificities that after the addition of rabbit serum as a source of complement, if the In the antigen-antibody reaction, the classical complement pathway is activated with the formation of the membrane attack complex that causes cell death.
This result is visualized microscopically with the use of vital stains and is a translation of the donor-recipient incompatibility, which invalidates the graft. Each reaction is evaluated for the percentage of dead cells and the results are recorded according to an internationally established system. 7
Organ transplantation is one of the areas where the study of histocompatibility antigens has the greatest impact. 8-10 The development of transplants in humans as altern