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Primary Antibodies

The primary antibody is the reagent that determines the test's specificity. 

Selecting a primary antibody is one of the key points in setting up an immunohistochemistry experiment. You may need to consider some or all of the following:-

Application

Not all primary antibodies will work in all applications. It cannot be assumed that an antibody used in an ELISA or for western blotting will also work in IHC, and vice versa. Check that the antibody is recommended for IHC – If further experiments are planned with other techniques, is there an antibody available suitable for all these methods?

Tissue Fixation

Because of the conformational changes that can occur during tissue processing, a primary antibody may not detect the conformation of the target protein after formalin fixation, or may not recognise the unprocessed format, so it’s important to match the antibody to the tissue fixation used on your sample.

Sometimes unmasking of your sections may be required to reveal the antigen and enable the antibody to bind to the antigen – make sure to include this step if it’s required in your method plan.

When multiple labelling, the antibodies will all need to be effective on the processed sample, although there can be some flexibility in mixing antibodies that need unmasking or not.

Specificity

Antibodies will have a greater affinity for the target the more closely they match the antigen. There may be related antigen proteins that share some sequence homology which could result in background staining.

Some antibodies are raised against specific termini, or fragments of the target protein that may offer a more specific signal, it’s important to ensure that the target sequence selected will be available to bind in your sample.

With highly conserved proteins, an antibody may be suitable to detect the protein in a range of different species. With proteins that have evolved more recently, there is less likelihood that the antibody will recognise a similar protein from a different species.

Clonality

Polyclonal antibodies are most often ‘neat’ serum from an immunised animal. There could be a wide range of Ig types, and a range of specificities for the antigen in question, depending on the immunogen used. Although this broader spectrum can be useful in some cases and can tolerate more minor differences in the antigen structure, polyclonals are more likely to bind off target as they may detect part of the antigen that has some homology with other proteins.

Monoclonal antibodies are derived from a single clone, so there is not the same variation of the  binding site of the antibody to the target protein as seen with a polyclonal antibody. Only the clone producing antibody specific to the immunogen should have been selected.  This results in a better signal to noise ratio in the final staining, but these antibodies often need to be used more concentrated than a polyclonal or the method sensitivity may need to be increased; fewer primary antibodies can bind to each target, since they should all only recognise the same sequence .  This also applies to recombinant monoclonals.   

Antibody Purity

Affinity purified antibodies will likely have less off-target binding, but not all affinity binding is the same! Protein G or Protein A is often used to extract the Ig to purify serum or cell solutions, and will increase Ig concentration, but it is not increasing the specificity of the antibody, this would only be achieved by purification using the immunogen to positively select for specificity to the target protein. This purification of the antibody is more of concern for polyclonal antibodies, since the specificity of a monoclonal or recombinant should be very similar if not the same.

Tissue Species

If the antibody host is the same as the tissue species from which the sample is derived, or closely related it causes cross reactivity issues where the detecting antibody will also recognise endogenous Ig in the tissue, not just the primary. There can be ways to mitigate the issue with careful selection of the detection method, but a conjugated primary antibody may be required.   

If working in xenografts, cross reactivity to both species of tissue should be considered, both for the primary antibody and the detection reagents.

Conjugates

Most IHC is done with unconjugated primary antibodies, as conjugation can affect the performance of the primary antibody. However, there are some experiments where a conjugated primary is either required for cross reactivity issues, or the antigen is so abundant that the signal will be adequate with a direct detection method. There may be experiments to run in parallel where a directly conjugated primary is required or could shorten the protocol time. Some manufacturers will offer the same clone with a number of different conjugates available.

It’s possible to conjugate proteins in house, and there are a number of kits available on the market, however kits offering 100% of the protein recovery will often leave the labelling reagent in solution that is reported to interfere with the cleanliness of the following staining, or post conjugation clean up steps yield reagents with cleaner results, but will result in the loss of some protein.  The labelling reaction often targets amines for conjugation, even if these happen to be in the antigen binding site, thus inactivating the antibody. As primary antibodies are normally expensive, and minimum quantities may be required for labelling, being able to buy conjugates can save a lot of time, effort and grant!

Controls

Positive & negative controls are always recommended, both to work up a method and to check the background that might be present.

Some companies offer control blocks demonstrating a range of signal strengths to help aid scoring. Vector Laboratories have an excellent webinar that discusses appropriate controls for IHC. 

Validation

Checking that the antibody is conforming to the staining patterns expected, and searching the literature to confirm this can be time consuming, although a very necessary part of ensuring your results are true. Further information on validation can be found here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813849/

https://youtu.be/gWTXDl8zMuQ

The Human Protein Atlas is a useful for a consolidated view of the protein of interest and may help elucidate appropriate control materials. 

Choosing your antibody:-

The manufacturer should offer much of the information required as part of the product data sheet – The applications it’s been tested in, with recommended working dilutions, the unmasking required, any known cross reactivity between species, the immunogen used to elicit antibody production and references for validation. They may helpfully highlight ranges that have had further testing in house, such as the IHC-plus™ and PathPlus™ ranges from LSBio, and HistoSure from Synaptic Systems.

Good manufacturers will also be able to offer technical support in the unlikely event you struggle to get the expected appropriate staining. Companies prepared to investigate can help you get the best results and discover the root cause which can then be tackled more effectively.

All Primary Antibodies

Manufacturer validated collections
PathPlus Antibodies
IHC-Plus Antibodies
HistoSure Antibodies