Transcript Chemiluminecent microparticle MEIA)) immunoassay

• 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.
• 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.
Techno-logy Solid phase
Separation
Label
step
MEIA
Detection
technology
Latex
Glass fiber
Alkaline
Fluorscence
microparticle
matrix
phosphatase
detector
enzyme
CMIA
Magnetic
micriparticle
magnet
Chemiluminescent Chemiluninescence
compound
photomultiplier
tube
• 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.
• 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.
• 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.
1. 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.
2. 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.