IMMUNOLOGY TEXTBOOK - Mercury Podillia 2013

IMMUNOLOGICAL RESEARCH METHODS

Lymphocytotoxic test

To date, in most laboratories, HLA-A, B, C, and DR Antigens are determined using serological Methods, specifically the lymphocytotoxic test. This test is based on the ability of anti-HLA Antibodies, in the presence of Complement, to destroy lymphocytes bearing the corresponding antigenic determinants. Cell death is demonstrated by adding trypan blue. Consequently, dead, damaged Cells take up the stain, and their number is counted under a Microscope.

The microlymphocytotoxic test is a Modification of the lymphocytotoxic test. It requires as little as 1 µL of typing sera and a small number of cells. The microlymphocytotoxic test is standard and used in all typing laboratories worldwide. The panel of typing sera (typing panel) is created through research on serum samples containing anti-HLA antibodies. These antibodies can be induced during Pregnancy, upon Blood transfusions, or As a result of allograft transplantation. The primary producers of typing sera are multiparous women who become immunized against paternal HLA antigens during gestation.

To detect classical HLA antigens of the A, B, C, and DR loci, special antisera containing antibodies against these antigens are used. The two most common types of microlymphocytotoxic serological reactions for serological HLA typing are: 1) HLA typing on Terasaki plates using the complement-dependent microlymphocytotoxic assay (CDC), and 2) detection of HLA antigens by ELISA using Lambda Monoclonal Trays.

1) The Lambda Cell Tray™ (LCT™) complement-dependent microlymphocytotoxic test is designed for serum screening for the presence of Class I and class II HLA antibodies using a complement-dependent lymphocytotoxic assay. Lambda Cell Tray™ kits 30T, 60T, and 72T are intended for the screening and determination of class I HLA antibody Specificity. The Lambda Cell Tray™ 60B kit is designed for determining class II HLA specificity.

Principle of the method. Lymphocytes bearing known surface antigens are incubated with a serum sample and rabbit complement. If the serum contains specific antibodies against these antigens, cell lysis is triggered. If the test serum contains HLA antibodies, they can thus be detected.

The LCT™ Lambda Cell Tray™ is a frozen cell tray designed for PRA level determination and HLA antibody screening. LCT™ offers a diverse cell tray for the cytotoxic screening of antibodies in human serum. The trays consist of selected lymphocytes frozen within a Terasaki plate.

2) The ELISA test using Lambda Antigen Tray™ (LAT) plates is designed to detect HLA-specific antibodies in pre- and post-transplant recipients. It is characterized by high specificity, as LAT™ kits utilize purified class I and class II HLA antigens. The LAT™ kit recognizes both cytotoxic and non-complement-fixing antibodies, distinguishes between class I and class II HLA specificities, detects IgG and IgM antibodies, and includes antigens of rare HLA alleles.

THE PRINCIPLE OF the LAT™ method relies on pre-calibrated ELISA Reagents for the detection of IgG antibodies against class I and class II HLA molecules in human serum. Specific amounts of HLA antigens, purified by Affinity Chromatography, are coated into various wells of a Terasaki plate. Specific binding of antibodies from the test sample to any of these antigens is detected using alkaline phosphatase-conjugated antibodies that recognize exclusively human IgG. Quantitative measurements of the reaction degree are determined spectrophotometrically after The addition of an appropriate chromogenic substrate. Qualitative determinations of antibody specificity are assessed by analyzing the LAT™ reactivity map using the corresponding LAT™ work sheets.

LAT™ Lambda Antigen Trays™ are purified complexes of class I and class II HLA coated onto Terasaki plates, intended for the detection of HLA IgG antibodies. Upon the addition of patient serum, antibodies bind to the purified HLA antigens. The panel of HLA antigens allows for the detection of both complement-fixing and non-complement-fixing IgG antibodies.

The LAT™ Mixed method utilizing mixed-antigen plates is a preliminary HLA antibody screening method that allows multiple samples to be tested on a single tray. LAT™ Mixed plates contain a defined set of HLA antigens, including rare ones, and recognize both cytotoxic and non-complement-fixing antibodies.

The LAT™ Single Antigen method is an assay for detecting and typing HLA antibodies in patients with high PRA levels. All wells of the Terasaki plate for LAT™ Single Antigen are coated with single, distinct antigen preparations. LAT™ Single Antigen detects low-titer antibodies that might otherwise remain undetected by other methods in the presence of antibodies against more abundantly represented antigens. Using a panel of purified HLA antigens, the specificities of antibodies in sera with high PRA levels can be accurately identified.