Monoclonal primary

Basic protocol for multiple immunostaining using monoclonal primary antibodies of different IgG isotypes... 

Incubate specific mouse monoclonal primary antibody with FITC -conjugated mouse IgG specific Fab fragments at a ratio of 1 2 (weight for weight, based on concentration data supplied by manufacturers) in a small volume (e.g., in 10 pi or more, typically 1 pg of primary antibody in 10 pi) of staining buffer in a microcentrifuge tube for 20 30 min at room temperature. 

Fig. 9.1 Immunolocalization of Adenosine Receptor A (FITC, green) in mouse myocardium using mouse monoclonal primary antibody after blocking mouse endogenous immunoglobulins by preincubation with unconjugated Fab fragment Goat Antimouse IgG. Red color accounts for cardiomyocytes and erythrocytes autofluorescence captured under illumination with a filter excit ing the autofluorescence in red spectrum. Nuclei are counterstained with DAPI (blue). Courtesy of Stephanie Grote...

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

For monoclonal primary antibodies, nonspecific negative reagent controls may be developed by different methods. The optimal method is an antibody of the same isotype, present in the same immunoglobulin concentration, using the same diluent and exhibiting no specific reactivity with the given human tissues tested. A less optimal alternative is to use mixtures of antibodies representing all or most relevant IgG subtypes. Finally, the diluent itself may also be used as an alternative which, however, is neither efficient nor desirable. 

Dissociation of primary antibody during washing or incubation with link antibodies A feature of low affinity antibodies Polyclonal primary antiserum Attempt staining at low dilutions. Monoclonal primary antibody Replace with higher affinity antibody of identical specificity. Re-optimize incubation times for washing buffer and link antibody. 5-6... 

The simultaneous localization of antigens may be complicated if monoclonal primary antibodies are used for both epitopes. Monoclonal antibodies are often of the IgGl subclass, which cannot be distinguished by secondary antibodies. Hapten-labeling or biotin-avidin bridging could be of considerable value to circumvent this problem. 

Briefly, the procedure involves the sequential application of two different staining systems, both using a mouse monoclonal primary antibody. The first is an indirect ABCf peroxidase procedure, using biotinylated horse antimouse igC linked to ABC peroxidase, with AEC as the substrate. 

Lewis Carl SA, GiUete-Perguson 1, Ferguson DG (1993) An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies. J Histochem Cytochem 41 1273-1278. 

The secondary antibody will be one that recognizes the primary antibody, and to which alkaline phosphatase has been conjugated. The specific choice will depend on the nature of the primary antibody used in Subheading 3.2.3, step 4 thus, a mouse monoclonal primary antibody will dictate that the secondary antibody be an alkaline phosphatase-conjugated rabbit anti-mouse antibody. Typically, the secondary antibody may be used at a 1 10,000 dilution, although some empirical testing may be required. 

Somatostatin. Figure 1 Somatostatin-like im mu noreactivity in neurons of the periventricular hypothalamic nucleus of the rat. Coronal brain cryostat sections have been processed for im mu nohistochemistry and sequentially incubated with a primary monoclonal mouse anti human somatostatin antibody and secondary antimouse antibody conjugated with the fluorescence-dye Cy-3. Images have been taken with a Zeiss Axioplan fluorescence microscope. Scale bar, 100 pM. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

Korenaga, M., Hitoshi, Y., Takatsu, K. and Tada, I. (1994) Regulatory effect of anti-interleukin-5 monoclonal antibody on intestinal worm burden in a primary infection with Strongyloides venezuelensis. Journal of Parasitology 24, 951-957. 

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