Complement-dependent cytotoxicity (CDC) is an effector function of

IgG Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG a ...

and

IgM Immunoglobulin M (IgM) is one of several isotypes of antibody (also known as immunoglobulin) that are produced by vertebrates. IgM is the largest antibody, and it is the first antibody to appear in the response to initial exposure to an antig ...

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

. When they are bound to surface

antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

on target cell (e.g. bacterial or viral infected cell), the

classical complement pathway The classical complement pathway is one of three pathways which activate the complement system, which is part of the immune system. The classical complement pathway is initiated by antigen-antibody complexes with the antibody isotypes IgG and IgM ...

is triggered by bonding

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

C1q The complement component 1q (or simply C1q) is a protein complex involved in the complement system, which is part of the innate immune system. C1q together with C1r and C1s form the C1 complex. Antibodies of the adaptive immune system can bind ...

to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis. Complement system is efficiently activated by human IgG1, IgG3 and IgM antibodies, weakly by IgG2 antibodies and it is not activated by IgG4 antibodies. It is one mechanism of action by which

therapeutic antibodies Monoclonal antibody therapy is a form of immunotherapy that uses monoclonal antibodies (mAbs) to bind monospecifically to certain cells or proteins. The objective is that this treatment will stimulate the patient's immune system to attack those c ...

The Role of Complement in the Mechanism of Action of Rituximab for B-Cell Lymphoma: Implications for Therapy. Zhou 2008
/ref> or antibody fragments can achieve an antitumor effect.

Use of CDC assays

Therapeutic antibodies

Development of antitumor therapeutic antibodies involves ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

'' analysis 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 ( serum). 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 (live cells staining):'' after incubation of target cells with antibodies and complement,

plasma membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (t ...

-permeable dye is added (e.g.

calcein Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495/515 nm, respectively, and has the appearance of orange crystals. Calcein self- quenches at concentrations above 70mM ...

-AM or resazurin). Live cells metabolise it into impermeable

fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

product that can be detected by

flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...

. This product can’t be formed in metabolically inactive dead cells. * ''Measuring of the activity of released intracellular enzymes'': dead cells release

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...

(e.g. LDH or

GAPDH Glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) () is an enzyme of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. In addition to this long establishe ...

) and addition of its substrate leads to color change, that is usually quantified as change of

absorbance Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative lo ...

or luminiscence. * ''Dead cells staining:'' a (fluorescent) dye gets inside the dead cells through their damaged plasma membrane. For instance

propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...

binds to DNA of dead cells and fluorescent signal is measured by flow cytometry.

HLA typing and crossmatch test

CDC assays are used to find a suitable donor for organ or

bone marrow Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...

transplantation, namely donor with matching

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...

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 (other words serologic typing) uses batch of anti-HLA antibodies from characterised allogeneic

antisera Antiserum is a blood serum containing monoclonal or polyclonal antibodies that is used to spread passive immunity to many diseases via blood donation (plasmapheresis). For example, convalescent serum, passive antibody transfusion from a previous ...

monoclonal antibodies A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies ca ...

. These antibodies are incubated one by one with patient‘s or donor‘s

lymphocyte A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic ad ...

s and source of complement. Amount of dead cells (and thus positive result) is measured by dead or live cells staining. Nowadays CDC typing is being replaced by molecular typing, which can identify

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...

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 (positive test) 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 (AHG), removing unbound antibodies before adding complement, separation of

T cell A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell r ...

and

B cell B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or ...

subset. Besides CDC crossmatch there is flow-cytometric crossmatch available, that is more sensitive and can detect even complement non-activating antibodies.

References

{{reflist * Immunology

Use of CDC assays

Therapeutic antibodies

HLA typing and crossmatch test

See also

References