Antibody labelling

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. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Currently, there are several assays for the measurement of PSA. All of them contain monoclonal or polyclonal antibodies labeled with enzymatic, fluorometric, or radioactive markers. These assays have shown significant variations within the same patient specimens. These variations may result from differences in antibody specificity, reaction kinetics, calibration, or the system s sensitivity. Studies have shown that only free PSA and PSA-ACT show immunological reactivity in these assays. Also, reaction kinetics can influence the molar ratio. Some of these assays with shorter incubation times may specifically bind the free PSA molecule (which is a lower weight form of PSA). In the equimolar assays, changing the incubation... 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

However, problems are encountered in production of rhenium radionuclides and work is being done to increase the yields of the radionuclides to meet urgent demands for their use in therapy. Moreover, rhenium is not as reactive as technetium. This situation makes rhenium chemistry somewhat specific - optimum conditions in the preparation of rhenium complexes or in antibody labeling using bifunctional ligands must be identified. 

The level of TRITC modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic absorption maximum ( 575nm). The number of fluor-ochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is especially important in predicting the behavior of antibodies labeled with TRITC. For a TRITC-labeled protein, the ratio of its absorbance at 575-280 nm should be between 0.3 and 0.7. 

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

For other examples of antibody labeling protocols see Goding (1976) and Harlow and Lane (1988). 

Visualizing more than one epitope on one section can be accomplished by different fluorescence labeling or different sizes of colloidal gold coupled to primary or secondary antibodies. Primary antibodies from different species and adequate secondary antibodies labeled differently can be used. In case of primary antibodies from the same species, the hapten technique can be applied. A hapten is a small molecule that can be bound to antibodies dinitrophenol and arsinilate are typically used as haptens. Again, adequate secondary antibodies labeled differently can be used (14,17,32). A collection of protocols for multiple immu-nolabeling has been described by Beesley (37). 

As an example of the use of antibodies labeled with alkaline phosphatase for detection of in situ hybridization, an infection with BNYVV virus in sugar beet is shown in Fig. 3C. Lectins labeled with an avidin-biotin fluorescein conjugate was used to visualize a-galactosyl groups on the surface of S. pombe in Fig. 3D. 

Immunoradiometric assays (IRMAs) are like RIAs in that a radiolabeled substance is used in an antibody-antigen reaction, except that the radioactive label is attached to the antibody instead of the hormone. Furthermore, excess of antibody, rather than limited quantity, is present in the assay. All the unknown antigen becomes bound in an IRMA rather than just a portion, as in a RIA IRMAs are more sensitive. In the one-site assay, the excess antibody that is not bound to the sample is removed by addition of a precipitating binder. In a two-site assay, a molecule with at least two antibody-binding sites is adsorbed onto a solid phase, to which one of the antibodies is attached. After binding to this antibody is completed, a second antibody labeled with 125I is added to the assay. This antibody reacts with the second antibody-binding site to form a sandwich, composed of antibody-hormone-labeled antibody. The amount of hormone present is proportional to the amount of radioactivity measured in the assay. 

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