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Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other
M J Watts1, J R Dankert, E P Morgan
1Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, U.K.
Abstract:
We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.
Insights
Researchers identified a membrane-attack-complex-inhibiting protein (MIP) in human body fluids. This protein, identical to the erythrocyte-derived MIP, plays a role in complement activation and erythrocyte lysis.
Area of Science:
- Immunology
- Complement system biology
Background:
- A membrane-attack-complex-inhibiting protein (MIP) was previously isolated from human erythrocyte membranes.
- Polyclonal antibodies against erythrocyte MIP were generated.
Purpose of the Study:
- To identify and characterize MIP in human body fluids.
- To develop a method for quantifying MIP in plasma, urine, and cerebrospinal fluid.
- To investigate the role of plasma MIP in complement activation.
Main Methods:
- Immunochemical identification of MIP in plasma, urine, saliva, and cerebrospinal fluid.
- Immunoaffinity chromatography for plasma MIP isolation.
- SDS/polyacrylamide-gel electrophoresis for molecular mass determination.
- Generation of monoclonal antibodies and development of a two-site enzyme-linked immunosorbent assay (ELISA).
Main Results:
- A protein immunochemically identical to erythrocyte MIP was found in plasma, urine, saliva, and cerebrospinal fluid.
- Plasma MIP has a molecular mass of 55 kDa (non-reduced) and 65 kDa (reduced).
- A sensitive ELISA was established for MIP quantification in various body fluids.
- Plasma MIP deposition on erythrocyte membranes during complement activation is C8-dependent.
- Depletion of plasma MIP enhanced hemolytic activity against heterologous erythrocytes.
Conclusions:
- MIP is present in various human body fluids and is immunochemically identical to the erythrocyte-derived protein.
- Plasma MIP is involved in the regulation of complement-mediated lysis.
- The developed ELISA allows for the quantification of MIP in clinical samples.