Multiple Antibodies Different Species

Table 11.2 Controls for indirect multiple antibodies different species Controls for indirect multiple antibodies different species...

Fig. 11.1 Multiple 1° antibodies different species, (a) Antigens Ag A and Ag B before incubations with antibodies, (b) The 1° antibody mouse anti-Ag A binds to Ag A and 1° antibody rabbit anti-Ag B binds to Ag B. (c) The 2° antibody goat anti-mouse 488 fluorophore binds to the mouse anti-Ag A and 2° antibody goat anti-rabbit 555 fluorophore binds to the rabbit anti-Ag B. The antibodies were combined in a single incubation for the 1° and the 2° antibodies...

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). 

Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species and accordingly can be visualized with differently labeled species-specific secondary antibodies (see Sect. 8.1). However, quite often the appropriate combination of primary antibodies from different host species is not available. A general problem relates to the fact that the available primary antibodies may originate only from one species either rabbit or mouse. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies (Ino 2004). 

As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. 8.2. and 8.3 below. 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

Immobilized lysate antigen of E. coli 0157 H7 at different concentrations was assayed and the low detection limit was 3 gg/mL. The assay also demonstrated very good specificity different microbial lysate antigens were immobilized, including E. coli and H. pylori, and the primary and secondary antibodies were mixtures of different species. The assay time is only 25 min (the conventional lab based assay requires over 3 h) the sample consumed was less than 12 pL. While still an un-optimized chip, this lA chip shows a great potential in detecting multiple pathogen efficiently. 

Multiple 1° antibodies used together in one experiment must be generated in different species of animals. 

There are two general strategies to use multiple 1° antibodies (1) 1° antibodies made in different species and (2) 1° antibodies made in the same species. Initially, it might appear that this difference is not worth separate chapters however, the important issue is how to handle the 2° antibodies. For experiments with 1° antibodies in different species, such as mouse and rabbit, 2° antibodies are made against the... 

Using 1° antibodies made in the different species is the easiest way to localize multiple proteins (Chapter 11). However, it is not possible to get all 1° antibodies needed from a different species. This is especially true because there are so many mouse monoclonal antibodies available. Eventually, an experiment will need two mouse 1° antibodies or two rabbit 1° antibodies. This chapter presents the concept of combining multiple 1° antibodies made in the same species of animals. Two different approaches include block-between method and labeled Fab procedure (Lewis et al., 1993) that is available as the commercial product, Zenon (Molecular Probes/Invitrogen). 

Multiple 1° antibodies from different species - a method with two 1° antibodies... 

All antibodies age and therefore have a specific shelf life. Aging may be different for different antibodies, and real aging may be quite different from the expiry dates printed on containers of antibodies. Mixtures of antibodies as are found in secondary antibody cocktails may show distinct aging differences. In other words, over time, one of the species in a secondary cocktail may age at a more rapid rate than the other(s). This would result in a significant decrease in sensitivity for that particular species of primary antibody. A user performing IHC stain runs with multiple tissues, and using primary antibodies from more than one species, must utilize primary controls for each species of primary antibody to detect a change in one of the components of the secondary antibody cocktail. 

The protocol for double/multiple immunolabeling using haptenylated primary antibodies is essentially the same as with primary antibodies of different IgG isotypes. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated. In this case, you have to carry out the immunostaining in two steps in the first step you visualize the unlabeled first primary antibody with a secondary species-specific antibody, and in the second step you can detect the second primary haptenylated antibody via another secondary antibody directed against the corresponding hapten. Should the hapten be a fluorophore, it can be visualized directly in a fluorescent microscope and you do not need the second step... 

They demo ns baled that the enzyme is immunologic ally cell and species specific. Reaction with the antibodies resulted in one precipitation line, indicating that the enzyme U immunologically homogeneous and that the different bands obtained after electrophoresis represent multiple forma of the enzyme. 

Disadvantages of indirect immunofluorescence include the potential for cross-reactivity and the need to find primary antibodies that are not raised in the same species or of different isotypes when performing multiple-labeling experiments. Samples with endogenous immunoglobulin may exhibit a high background. 

---