DOC

Roche ECL Priniciple

By Randall Cunningham,2014-05-03 09:39
10 views 0
Roche ECL Priniciple

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    ECL (IA) technology

    Introduction

    The advantage of electrically initiating the chemiluminescent reaction is that the

    entire reaction can be precisely controlled. E Electro

    C Chemo

    L Luminescence

     Immuno I

    A Assay

    The key elements and substances in the ECL process are: o Measuring cell

    o Voltage

    o Platinum electrode

    o Magnet

    o Photomultiplier

    o Antigen/antibody

    o Biotin

    o Paramagnetic microbeads coated with streptavidine o ProCell solution (TPA-Tripropylamine with a phosphate buffer)

    o CleanCell solution (KOH cleaning solution)

Test principles

    Sandwich principle

    o High molecular weight antigens are measured Examples: TSH, CA 15-3 II-tests

    Competitive principle

    o Low molecular weight antigens are measured Examples: T4, Folate II- tests

    Bridging principle

    o High molecular weight antibodies are measured Examples: Anti-HAV IgM, Anti-HBc IgM

The basic principle

The measuring cell

    The core of the detection unit is the flow-through ECL measuring cell.

    Three processes steps are performed in the cell: 1. Bound/free separation

    2. ECL reaction

    3. Release of microbeads and cell cleaning

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    The following figure shows the main components of the measuring cell: A Screw

    B Counter electrode

    C Optical window

    D Distance washer

    E Top cell

    F Cell gap

    G Gasket

    H O-ring

    I Diaphragm

    J Reference electrode

    K Outlet fitting

    L Working electrode

    M Movable magnet

    N Inlet fitting

    O Cell body

Bound/Free separation

    o Streptavidin microbeads coated with antigen-antibody complex are aspirated. o The magnet is activated.

    o The antigen-antibody complex is captured by the magnet onto the working electrode.

    o A TPA solution (ProCell) is aspirated to wash the microbeads on the working electrode.

    o TPA (ProCell) is used to flush out the excess reagent and sample material.

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

ECL reaction

    o The ruthenium complex and TPA (ProCell) are involved in the reactions. o They remain stable as long as no voltage is applied.

o Voltage is applied between the working and counting electrodes and an electrical

    field is created. 2+o An ECL reaction of ruthenium-tris (bipyridly) and TPA occurs on the surface

    of the electrode.

o TPA is oxidized at the electrode.

    o This releases an electron and forms an TPA radical-cat ion. +o This reacts by releasing a proton (H) to form a TPA radical (TPA*).

o The ruthenium complex also releases an electron.

    o The ruthenium complex then oxidizes to form the ruthenium cat-ion Ru(bpy) 33+ followed by the chemiluminescent reaction with the TPA radical.

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

o The ECL reaction is initiated.

    o Peak light emission occurs for a short time interval (0.20-0.60 s). o A photomultiplier detects and converts the ECL signal into an electric signal. o The corresponding signals are used for the calculation of results.

    Each measuring cycle requires a total of 42 seconds for the following two stages: Pre-conditioning 2 seconds (approximately)

    Bead capturing/BF separation 22 seconds (approximately)

    The magnet is deactivated before the measurement is started, to avoid any interference.

    Measuring ~ 2 seconds

    Cleaning ~ 14 seconds

    Re-conditioning ~ 2 seconds

    Release of microbeads and cell cleaning

    o Cleaning solution is aspirated to the measuring cell.

    o Microbeads are washed away from the electrode.

o TPA (ProCell) is aspirated.

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    o The surface of the measuring cell is regenerated by varying the electrode voltage. o The measuring cell is ready for another measurement.

    A Magnetic microbeads with bound antigen-antibody complex B Photomultiplier

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    C Counter electrode

    D Unbound antibody (ruthenium-labeled)

    E Flow channel

    F Magnet

    G Working electrode

    Advantages of ECL technology

    o Convenient liquid reagents are used, which are extremely stable (20?C on board up to 12 weeks).

    o A combination of enhanced sensitivity and short incubation times leads to high- quality assays and rapid results.

    o A large measuring range minimizes the need for dilutions and repeats, reducing handling time and reagent consumption.

    o Applicability of the technique to detect all analytes provides a solid platform for menu expansion.

Sandwich

    principle

    o This principle is applied

    to high molecular weight

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    antigens, such as TSH and CA 15-3 II.

    o The measurement is directly proportional to the sample concentration. Low signal = low concentration

    High signal = high concentration

    The sandwich principle is applied to higher molecular weight analytes, such as thyroid-stimulating hormone (TSH).

    In the first step, a patient sample is combined with a reagent containing biotinylated TSH antibody and a ruthenium-labeled TSH-specific antibody in an assay cup. During a nine-minute incubation step, antibodies capture the TSH present in the sample.

    In the second step, streptavidin-coated paramagnetic microbeads are added. During a second nine-minute incubation, the biotinylated antibody attaches to the streptavidin-coated surface of the microbeads.

    After the second incubation, the reaction mixture containing the immune complexes is transported into the measuring cell.

    The immune complexes are magnetically entrapped on the working electrode, but unbound reagent and sample are washed away by ProCell.

    In the ECL reaction, the conjugate is a ruthenium-based derivative and the chemiluminescent reaction is electrically stimulated to produce light. The amount of light produced is directly proportional to the amount of TSH in the sample.

    Evaluation and calculation of concentration of the antigen or analyte are performed by means of a calibration curve that was established using standards of known antigen concentration.

Competitive principle

    o This principle is applied to low molecular weight antigens., such as T4 and Folate II.

    o The measurement is directly proportional to the sample concentration. High signal = low concentration

    Low signal = high concentration

This principle is applied to

    analytes of low molecular

    weight, such as T3.

    In the first step, the sample and

    a specific anti-T3 antibody

    labeled with a ruthenium

    complex are combined in an

    assay cup.

    After the first incubation,

    biotinylated T3 and

    streptavidin-coated

    paramagnetic

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    microbeads are added.

    The still-free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten complex.

    The entire complex is bound to the microbeads through the interaction of biotin and streptavidin.

    After the second incubation, the reaction mixture containing the immune complexes is transported into the measuring cell.

    The immune complexes are magnetically entrapped on the working electrode, but unbound reagent and sample are washed away with ProCell.

    In the ECL reaction, the conjugate is a ruthenium-based derivative and the chemiluminescent reaction is electrically stimulated to produce light. The amount of light produced is indirectly proportional to the amount of antigen in the patient sample.

    The concentration of the antigen is evaluated and calculated by means of a calibration curve that was established using standards of known antigen concentration.

Bridging principle

    o This principle is applied to high molecular weight antigens, such as Anti-HAV IgM and Anti-HBc IgM.

    o The measurement is directly proportional to the sample concentration. Low signal = low concentration

    High signal = high concentration

The bridging principle is

    similar to the sandwich

    principle, except that the assay

    is designed to detect antibodies,

    not antigens (for example, IgG,

    Irma, and IgA).

    This is accomplished by

    including biotinylated and

    ruthenium-labeled antigens in

    the reagents for which the

    targeted antibody has affinity.

    In the first step, serum

    antibodies bind with the

    biotinylated and ruthenium-

    labeled antigens to form an

    immune complex.

    The immune complex then

    reacts with the biotinylated

    antigen.

    After the second incubation,

    the reaction mixture containing

    the immune complexes

    is transported into the measuring cell. The immune complexes are magnetically entrapped on the working electrode, but unbound reagent and sample are washed away by ProCell.

    In the ECL reaction, the conjugate is a ruthenium-based derivative and the chemiluminescent reaction is electrically stimulated to produce light.

更多精品文档请关注本人主页?可爱喜洋洋http:///lxn214237830;下载后将页眉去掉即

    可;

    The amount of light produced is directly proportional to the amount of analyte in the sample.

    The concentration of the antigen is evaluated and calculated by means of a calibration curve that was established using standards of known antibody concentrations. Assay protocols

    There are 28 test protocols or test steps that can be used on the system. These protocols are predefined by Roche Diagnostics for each test and cannot be changed by the operator.

    The sequence of the individual processes differs from test to test. The main important assay protocol numbers are shown below:

Legend:

    R1 = Reagent 1 R2 = Reagent 2

    RO = Universal diluent PS = Pretreatment solution (for assays such as

    B12, Folate II, and Anti-HBc)

    S = Sample/calibrator/control DL = Diluted sample

    B = Beads (microbeads in the assay reagent pack)

    i= Incubation D = Detection with magnet drive

Basic assay processing chart

    o Various pipetting steps, at least one incubation, and a measurement step. o At least three test components (sample, reagent, and microbeads) are pipetted into a cup.

    o After incubation, the reaction mixture is aspirated into the measuring cell where the measurement process takes place (a pipetting cycle is defined as 42 seconds). o The number of pipetting step/reaction mixture is dependent on the test method. o For some methods, predilution with diluent or pretreatment with a special reagent is necessary.

Report this document

For any questions or suggestions please email
cust-service@docsford.com