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Immunochemichal methods



The properties of high affinity and specificity of antibodies are successfully employed in assays used for quantification of specific analytes in complex mixtures. The method is indirect in the sense that it is the antibody-analyte complex that is detected rather than the analyte per se. However, if the quality of the antibody is good, i.e., low cross reactivity with other molecules, there is a strong relationship between the measured signal from the complex and the concentration of the analyte. This relationship can be determined by the use of calibrators with known concentrations, which allow for a standard curve to be constructed and the concentration in a sample to be calculated.


Enzyme linked immunosorbent assay (ELISA)

Direct ELISA
The principle of a direct ELISA is that the sample is adsorbed to a solid surface, e.g., a 96-well microtiter plate, followed by addition of the antibody. In the most basic setting the antibody is conjugated with an enzyme and the activity is directly proportional to the amount of bound antibody. The most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP) which both, depending on substrate, can catalyze reactions that give rise to different types of measurable signals such as luminescence or changes in absorbance or fluorescence.

Sandwich ELISA
In a sandwich ELISA a pair of antibodies, with different epitopes, is used. A higher specificity is achieved, compared with the direct ELISA, since two distinct binding sites must be present for an analyte to be detected, which decreases the risk of cross reactivity. The capture antibody is immobilized on a solid surface followed by the sequential addition of sample and the enzyme-conjugated detection antibody. The different detection systems are the same as for the direct ELISA.

The AlphaLisa is a sandwich assay in which the capture and detection antibodies are bound to different types of micro spheres called donor and acceptor beads. A short-lived reactive oxygen specie, singlet oxygen, is produced when the donor beads are irradiated with a laser. Light with another wave length is emitted if the singlet oxygen reaches an acceptor bead, which needs to be in close proximity. One major advantage with this method is that it is homogenous, i.e., there are no wash steps between the different additions, which reduce the time consumption and eliminate possible errors introduced by the washes.
For more information about AlphaLISA, see: Extern link

Multiplex Immunoassays

Different techniques are available for simultaneous measurements of more than one analyte and the platforms described below share the feature that they are built on the sandwich ELISA principle. The main gain in using these multiplex assays is that the time and sample consumption is decreased compared to when each analyte is assayed one by one.

The xMAP technology utilizes microspheres, filled with a mixture of two fluorophores, as the solid surface. The blend of the fluorophores is used to identify which capture antibody that is bound. A third fluorophore, conjugated to the detection antibody, is used as a reporter instead of an enzyme. With this method up to 100 different analytes can be measured simultaneously in a 96 well/plate format. xMAP is an open format in the sense that it is possible to buy components to make in house assays.
For more information about the xMAP technology, see: External link

Meso Scale
Up to 10 different analytes can be measured at the same time with the Meso Scale platform, which utilizes electrochemical luminescensce for detection. The format is 96 well/plate and the capture antibodies are adsorbed at predefined locations in the wells in order to discriminate between the different analytes. It is not possible to make in house multiplex assays using this platform.
For more information about Meso Scale, see: External link

Randox distribute the capture antibodies on pre defined locations on a membrane using their own format. The detection antibody is conjugated with HRP that catalyze a light-emitting reaction. A maximum of 25 analytes can simultaneously be measured.
For more information about Randox, see: External link



Sidansvarig: Staffan Persson|Sidan uppdaterades: 2010-06-16

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