Immunoglobulins labeling with

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Fig. 4. Testing of a conjugate prepared from purified antibodies to mouse immunoglobulin labeled with alkaline phosphatase. Ordinate conjugate dilution abscissa absorbance at 405 nm of samples diluted 1 4 after 15 min of incubation with substrate. — , Dose response in microtiter wells coated with mouse serum proteins O—O, dose response in uncoated wells.

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

M. Okochi, H. Ohta, T. Tanaka, and T. Matsunaga, Electrochemical probe for on-chip type flow immunoassay immunoglobulin G labeled with ferrocenecarboaldehyde. Biotechnol. Bioeng. 90, 14-19 (2005). 

With this technique, the often numerous primary antibodies used do not all have to be labeled with enzyme, provided they are all of the same species that a secondary antibody raised against immunoglobulin will recognize. The secondary antibody, labeled with enzyme, provides for the detection of all of the primary antibodies without the need for them to be individually conjugated. The problems of background and denaturation associated with conjugation are still present and can be troublesome. However, an increment of sensitivity can be achieved greater than that obtained by the previous method. 

A secondary antibody labeled with biotin, which is reactive against the species of immunoglobulin used for the primary reagent. 

The use of antibodies labelled with a fluorescent derivative obtained from fluorescein, rhodamine or other fluorophore is well suited for the measurement of human or animal immunoglobulins. However, the same principle is not yet used for simple organic molecules. Discrimination between the labelled and non-labelled species at the isolation stage can occur for small organic molecules. However, there are a few examples of assays in which isolation is followed by fluorescence measurement. 

One of the earliest efforts of qualitative measurement of a protein (human serum albumin) in a microchip-based device was based on bead agglutination in a microchamber (approximately lOjaL). Subsequently, several quantitative immunoassays have been performed using microchip electrophoretic systems that permit separation and quantitation of free- and bound-labeled antigens in competitive assays (see Chapter 5). Most are carried out in channels micro-machined into fused silica substrates. Early work on quantitative assays achieved measurement of cortisol in serum.The assay used cortisol labeled with fluorescein and an argon laser detector at 488 nm and required only 80 pL of a 40x dilution of serum as the sample. Other capillary electrophoresis-based assays for a variety of antibodies have also been developed that include immunoglobulins (IgG, IgA, and IgM), antibovine serum albumin, and antiestradiol. ... 

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