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...

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

Steps in a two 1° antibody different species indirect immunocytochemistry experiment. 

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). 

Basic protocol for simultaneous immunoenzymatic double staining using primary antibodies of two different species or two different IgG isotypes... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted primary antibodies raised in two different species (e.g., rabbit and mouse) or belonging to two different IgG isotypes (e.g., IgGl and IgG3). Wash sections in PBS or TBS for 3 x 3 min. 

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. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Since antibodies of different species and different subclasses are variably cleaved by pepsin and papain, a test run is recommended to check incubation time and cleavage conditions. Take samples of different incubation time, freeze rapidly, and monitor the fragmentation by SDS-PAGE at the end of the experiment. Apply the samples both with and without DDT. 

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]. 

Not all proteins bind to antibodies in the usual binding cavity. Protein G, a cell surface protein from Streptococcus bonds to IgG molecules from many different species. Its binding site is on the outer surface of the heavy chain CH1 domain.96... 

For best results, plates should be coated with the IgG fraction of an antiserum, this can be conveniently prepared using caprylic acid precipitation [see Chapter 10 and ref. 15). Where this is not possible, indirect capture may be used, such as antispecies antibody on the plate, or the streptavidin-biotin system. Any indirect capture system must be compatible with the final label, e.g, labeled antigen or different species antisera with no crossreaction with die indirect coating antibody 11. Safety data (from ref. 16)... 

If no staining is observed in the positive control, check whether a reagent has been inadvertently omitted or whether the wrong reagent has been used (for example, antimouse Ig on a rat monoclonal). This is easy to do if several different antibodies from different species are being used. 

One of the most likely reasons for failure with this technique is the wrong choice of antibodies. It is important that the two primary antibodies have been raised in different species and that the two secondary antibodies have been raised in species distinct from either of the primary antibody species... 

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