Why choose multiple antibodies for IHC and IF experiments

We all know that when preparing IHC/IF samples, especially in the process of paraffin embedding, chemical and physical treatments may destroy or block certain epitopes in the target protein (antigen). As a result, monoclonal antibodies that can only recognize a single epitope cannot detect a specific antigen. Conversely, since multiple antibodies can recognize multiple epitopes on one antigen, they can still recognize the target protein by binding to other epitopes.

High-quality multi-strain antibodies are composed of multiple antibodies that identify epitope at different positions on the antigen; therefore, when performing IHC / IF analysis, each antigen will be connected to multiple antibodies. This feature can increase sensitivity and enhance the signal.

In addition, proteins in nature have many different variants. If epitope is presented in a different way, it may not be recognized by monoclonal antibodies, which will affect protein detection. However, multiple antibodies can recognize multiple epitopes to prevent them from being affected by variants. Based on the above reasons, many scientists prefer to choose multiple antibodies rather than monoclonal antibodies when selecting IHC/IF antibodies due to their high sensitivity and resistance to variants.

Formalin-fixed and paraffin embedded rat brain labeled with Rabbit Anti phospho-HSF1 (Ser326) Polyclonal Antibody (bs-3741R) followed by conjugation to the secondary antibody and DAB staining
Formalin-fixed and paraffin embedded rat colon labeled with Anti-CD8 alpha Polyclonal Antibody (bs-4791R), followed by secondary antibody was Goat Anti-Rabbit lgG, Cy3 conjugated (bs-0295G-Cy3). DAPI was used to stain the cell nuclei.

Although multiple antibodies have the above advantages, there are still many monoclonal antibodies that perform well in IHC/IF applications. Therefore, when deciding whether to use a monoclonal antibody or a multi-strain antibody, the basic considerations are still based on the experimental conditions and purpose.

Below we summarize some of the advantages and disadvantages of each antibody:

Multiple antibodies
(identify multiple epitopes)
Monoclonal antibody
(identify a single epitope)
advantage
High sensitivity High specificity
Enhanced signal Reduce cross reactivity (Cross reactivity)
Even if the protein is denatured or changed in configuration, it can still perform well If you need to quantify protein expression, monoclonal antibodies can get better results
High stability, can withstand changes in pH or buffer Small batch difference
Disadvantage
Big batch difference If the protein is denatured or changed, it may not be detected
High probability of cross reactivity Less suitable for applications that require rapid binding to the target protein
Sensitive to pH and buffer changes

Why are multi-strain antibodies better tolerant to pH and buffer changes than single-strain antibodies?

Generally, the difference in stability between multi-strain antibodies and monoclonal antibodies is due to their immunoglobulins Biophysical diversity.
Monoclonal antibodies are identical immunoglobulins produced by a single B cell.
Therefore, these same immunoglobulins have the same PTM and isoelectric point,
and only work well under optimal conditions (PH and Ion strength).

Multi-strain antibodies are a mixture of multiple monoclonal antibodies produced by different B cells.
Each monoclonal antibody in the mixture has its own unique PTM and isoelectric point.
Even if the experimental conditions (PH or Ion strength) are not optimized
, some of the immunoglobulins (monoclonal antibodies)
of the multi-strain antibodies may still work well and show the “stable properties” of the multi-strain antibodies.

 

 

 

 

We all know that when preparing IHC/IF samples, especially in the process of paraffin embedding, chemical and physical treatments may destroy or block certain epitopes in the target protein (antigen). As a result, monoclonal antibodies that can only recognize a single epitope cannot detect a specific antigen. Conversely, since multiple antibodies can recognize multiple epitopes on one…

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