You’ll give me an (enzymatic) complex!
Following on from Part 1 of this article, let’s start by having a look at the two most popular enzymatic ‘sandwich’ methods;
- The Peroxidase anti Peroxidase method (PAP). The PAP method was the first sandwich method that I used and involves three main stages- application of primary antibody, secondary antibody and PAP complex. Sandwich methods are still used extensively today, although the PAP method has fallen out of favour. The PAP is an antibody-HRP complex which increases the amount of enzyme (HRP) which can be used to detect a primary antibody. This gives it greater sensitivity than the direct and indirect methods and is used extensively in diagnostic immunohistochemistry. However, it’s a slightly inflexible method as only HRP substrates such as DAB (Brown) or AEC (Red) can be utilised.
- The Alkaline phosphatase anti Alkaline Phosphatase method (APPAP). APPAP is a companion to the PAP method which allows the use of different alkaline phosphatase substrates and therefore different coloured end points. In both the PAP and the APAAP the secondary is a link between the primary and the enzyme complex. For example if the primary was rabbit, the PAP complex would also be raised from rabbit, the linker would then be anti-rabbit (e.g. goat anti rabbit).
In their day the PAP and APAAP detections were very sensitive. To push the sensitivity of the detection further, researchers looked at different ways to get more enzyme in the detection system to increase the intensity of the coloured substrate reaction. This included repeated application of the detection system by re-applying the linker and PAP (or APAAP) complex several times, and although this could improve sensitivity, background levels also tended to increase. Another modification was to use a labelled secondary and then the PAP or APAAP complex, again with the sole aim of getting more enzyme into the detection system.
A step ahead of your competition
In my opinion gaining an understanding of different detection systems and how they can be applied can give your research an advantage over your competition. By understanding the principles you can apply or adapt detection systems to meet your immediate aims or the reagents that are available in your lab.
Although you may never use a PAP method in your research you will probably use a sandwich method with modern polymer detections. Often these are two step procedures e.g. primary followed by polymer, their flexibility and versatility can be improved by using the appropriate linking antibody (assuming you know what species proprietary polymers are raised in).
Understand the ingredients…
Sensitivity does not only come from the antibody reagents, choice of fluorochrome and substrate can also contribute to sensitivity, as does fixation, tissue preparation and antigen retrieval. It’s a bit like cooking- by understanding your ingredients and recipes you can adapt and modify to produce different dishes or compensate when an ingredient is missing.
…to make your sandwiches more tasty!
The classic cheese sandwich recipe (bread/cheese/bread) and the classic immuno sandwich (rabbit/goat/rabbit or primary/link/tertiary) are pretty similar but if you understand the recipe or principles and can use other ingredients or reagents then your options become unlimited.