Chemiluminecent microparticle)) immunoassay

Chemiluminecent microparticle immunoassay Chemiluminecent microparticle immunoassay CMIA used for quantitative determination of human thyroid stimulating hormone in serum and plasma. In ARCHITECT instrument, TSH assay is a two-step immunoassay to determine the presence of TSH in serum or plasma by CMIA technology with flexible assay protocols referred to as chemiflex.

In the first step, sample, anti TSH antibody coated paramagnetic micro particles and TSH assay diluent are combined. TSH present in the sample binds to the anti TSH antibody coated micro particles. After washing, anti- alpha TSH acridinium labeled conjugate is added in the second step. Pre-trigger and trigger solutions are then added to the reaction mixture: the resulting chemiluminescent reaction is measured as relative light units(RLUs).

A direct relationship exists between the amount of TSH in the sample and the RLUs detected by the instrument optical system.

Microparticle Enzyme Immunoassay (MEIA) is an immunoassay method that utilizes the isolation of antibody/antigen complexes on a solid phase surface of small beads called microparticles. MEIA has been widely adapted to automate the measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis, and thyroid testing.

Assay components The components of MEIA include the following, all suspended in a specific buffer optimized for the assay: Microparticle-Antibody Solid Phase: Latex microparticles that are coated with antibody to bind the specific analyte being measured; •Antibody-Enzyme Conjugate: Alkaline Phosphatase enzyme conjugated to antibody; •Enzyme Substrate: Fluorescent 4-Methyl Umbelliferone Phosphate (MUP) in solution that is available for a reaction with the enzyme on the antibody.

Assay principle MEIA: technology uses a solution of suspended, submicron sized latex particles to measure analytes. The particles are coated with capture molecule specific for the analyte being measured. The effective surface area of micro particles increases assay incubation time. This permits MEIA assays to be competed in less time than other immunoassay.

In the sampling center, reactants and sample for one assay are transferred to a reaction vessel. The reaction vessel is transferred to the processing center where reagents and sample are incubated to allow them to come to reaction temperature. The reagents and sample are combined and the reaction mixture is transferred to an inert glass fiber matrix. Irreversible binding of the microparticles causes the immune complex to be retained by glass fibers while the reaction mixture flows rapidly through the large pores in the matrix.

An alkaline phosphate- labeled conjugate is added to the glass fiber matrix prior to the addition of 4- methylumbelliferyl phosphate MUP. The conjugate catalyzes the hydrolysis of MUP to methylumbelliferone(MU). Measurement of the fluorescent MU as it is generated on the matrix is proportional to the concentration of the analyte in the test sample.

MEIA reaction sequences There are two types of reaction sequences or formats for MEIA assays: One step: Sample, microparticles and conjugate are combined in the incubation well of the reaction vessel. Two step: Sample and microparticles are combined in the incubation well of the reaction vessel and the conjugate reaction takes place on the matrix cell.

Procedure Analytes bind to micro particles: Samples and micro particles are combined and incubated at reaction temperature. During the incubation period analytes bind to the micro particles creating an immune complex. Immune complex binds to glass fiber matrix. The processing probe aspirates the reaction vessel and dispenses it onto the matrix cell. The immune complex binds irreversibly to glass fiber matrix. A matrix cell wash removes unbound materials. The immune complex is retained by the glass fibers while the excess reaction mixture flows rapidly through the large ores in the matrix.

MEIA magnet CMIA Detection technology Label Separation step Solid phase Techno-logy Fluorscence detector Alkaline phosphatase enzyme Glass fiber matrix Latex microparticle MEIA Chemiluninescence photomultiplier tube Chemiluminescent compound magnet Magnetic micriparticle CMIA