Development of antitumor therapeutic antibodies involves in vitroanalysis of their effector functions including ability to trigger CDC to kill target cells. Classical approach is to incubate antibodies with target cells and source of complement. Then cell death is determined with several approaches:
Radioactive method: target cells are labeled with 51Cr before CDC assay, chromium is released during cell lysis and amount of radioactivity is measured.
Measuring of the metabolic activity of live cells : after incubation of target cells with antibodies and complement, plasma membrane-permeable dye is added. Live cells metabolise it into impermeable fluorescent product that can be detected by flow cytometry. This product can’t be formed in metabolically inactive dead cells.
Measuring of the activity of released intracellular enzymes: dead cells release enzyme and addition of its substrate leads to color change, that is usually quantified as change of absorbance or luminiscence.
CDC assays are used to find a suitable donor for organ or bone marrow transplantation, namely donor with matching phenotype of histocompatibility system HLA. At first, HLA typing is done for patient and donor to determine their HLA phenotypes. When potentially suitable couple is found, crossmatch test is done to exclude that patient produces donor-specific anti-HLA antibodies, which could cause graft rejection. CDC form of HLA typing uses batch of anti-HLA antibodies from characterised allogeneicantisera or monoclonal antibodies. These antibodies are incubated one by one with patient‘s or donor‘s lymphocytes and source of complement. Amount of dead cells is measured by dead or live cells staining. Nowadays CDC typing is being replaced by molecular typing, which can identify nucleotide sequences of HLA molecules via PCR. CDC assay is usually used for performing crossmatch test. The basic version involves incubation of patient’s serum with donor’s lymphocytes and second incubation after adding rabbit complement. Presence of dead cell means that donor isn‘t suitable for this particular patient. There are modifications available to increase test sensitivity including extension of minimal incubation time, adding antihuman globulin, removing unbound antibodies before adding complement, separation of T cell and B cell subset. Besides CDC crossmatch there is flow-cytometric crossmatch available, that is more sensitive and can detect even complement non-activating antibodies.
