Antibody Resource Guide

Understanding what is the difference between monoclonal and polyclonal antibodies can be difficult. We’ve created this antibody resource guide to outline how these antibodies differ in the way they are produced and what they can be used for. Below is a breakdown outlining everything you need to know about polyclonal and monoclonal antibodies.

 

Key differences between Polyclonal and Monoclonal Antibodies:

Polyclonal
Monoclonal
Heterogeneous population of antibodies with differing paratopes for an antigen
Homogenous population of a specific antibody with one paratope
Not Epitope Specific
Epitope Specific
Increased likelihood for cross-reactivity with similar antigens Low cross-reactivity
Increased likelihood for background noise Low background noise
Lot Variability Identical lots
Inexpensive to develop Expensive to develop
Quick to produce (approx. 3 months) Slow to produce (approx. 6 months)
Many host species options Few host species options

What are Monoclonal Antibodies?

Monoclonal AntibodiesMonoclonal Antibodies Definition:

Monoclonal antibodies consist of one (mono) type of antibody that binds to a specific epitope on the target antigen

Monoclonal Antibody Production

Monoclonal antibodies are created by first injecting an immunogen into a host animal. After the immunogen has caused an immune response, the B-cells from the spleen are removed and fused with myeloma cells (cancer B-cells).

This is done to create an immortal cell line, as healthy spleen cells would not survive indefinitely in cell culture. Through this process a hybridoma cell line is created.

MiceSingle B-cells from the spleen are fused and cultured into their own hybridomas, therefore creating a mono-culture of B-cells all producing the same specific antibody.

Each hybridoma now producing a monoclonal antibody, is then assigned a specific clone number in order to identify the particular antibody and the specific epitope that it binds.

The monoclonal antibodies are excreted by the B-cells into the cell culture media, where they can be extracted for further testing and purification.

Once a monoclonal antibody hybridoma clone is created, it is a stable renewable source of antibodies. This ensures that each batch is identical to the previous one.

Hybridomas can also be injected in the peritoneal cavity of a mouse in order to create a tumor that secrets a fluid rich in antibodies called ascites fluid.

 

Best uses of monoclonal antibodies:

  • detecting a specific antigen
  • detecting a single member of a protein family
  • creating consistent results between experiments/batches
  • staining cells with less background – great for immunohistochemistry, immunocytochemistry, and immunofluorescence experiments
  • quantifying protein expression (ex. Flow cytometry or Fluorescence-activated cell sorting)
  • detecting changes in molecular conformation
  • detecting changes in phosphorylation states
  • detecting a target for X-ray crystallography
  • creating animal models lacking a specific cell type
  • immunotherapy

 

Monoclonal antibodies are not recommended for:

  • detecting target proteins across different species based on homology
  • detecting low levels of a target proteins
  • detecting a protein in an altered conformation

 

What are Polyclonal Antibodies?

Polyclonal AntibodiesPolyclonal Antibody Definition
Polyclonal antibodies consists of many (poly) different antibodies that bind to many different epitopes on the target antigen.

Polyclonal Antibody Production

Polyclonal antibodies are created by injecting an immunogen into a host animal. This immunogen causes an immune response in the animal and the activation of multiple B-cells which all target a specific epitope on the immunogen.

rabbitThis results in a large number of antibodies with varying paratopes, and therefore varying affinities for the target protein.

After immunization, these polyclonal antibodies can be used straight from the serum (blood which has had clotting proteins and red blood cells removed) or purified to obtain a solution which is free from other serum proteins.

Batch to batch variability is high as new animals will need to be immunized with the antigen to create a new batch.

 

Best uses of polyclonal antibodies:

  • to detect a known or unknown isoforms of antigens with high antigen homology
  • to detect low levels of a particular antigen
  • to capture as much antigen as possible (ex. Immunoprecipitation or Chromatin Immunoprecipitation)
  • to detect denatured proteins
  • to detect targets with possible genetic polymorphisms, glycosylation or conformational changes
  • to detect a native protein across multiple assay types
  • to detect a target in solutions with varying pH and salt concentrations

 

Polyclonal antibodies are not recommended for:

  • quantification experiments such as flow cytometry as the amplification of the signal due to the multiple binding sites will cause incorrect results
  • cases where cross-reactivity with high homology proteins is problematic

StressMarq offers a broad range of both monoclonal and polyclonal antibodies to allow researchers the flexibility to choose the best fit for their experiment.

If you need help deciding which antibody is right for your experiment, the scientists at StressMarq would be more than happy to assist you!

Just email us at [email protected] with your questions.