Antibodies in Practice: Proper Controls

Proper Controls

The use of proper controls will help eliminate any false positive and false negative results and will enable better interpretation of the experimental data. The key to proving antibody specificity is often the correct use of controls. They will also be invaluable in troubleshooting throughout the experimental design process. Here are some considerations:

  • Whenever possible, both negative and positive controls should be included in an assay.
  • A positive control sample may be any tissue, cell line, or purified protein that is known to contain the antigen of interest, and has been previously demonstrated to be positive by a reliable method.
  • A negative control sample is one that is known to be devoid of the antigen of interest. A cell line or tissue that is known not to express the protein of interest is a better negative control.
  • In addition to sample controls, one should also use reagent controls.
  • Remember to change only one experimental variable at a time.
  • One should run separate controls for primary and secondary antibodies.
  • Because antibodies from different animal bleeds or purification batches may have significantly different titer values, each new batch of antibody must be validated, and conditions optimized before use in an existing assay.

It should also be noted that an integral part of good laboratory practice is to keep complete documentation of all dilutions, diluents, incubation times, lot numbers, preparation dates of all reagents, and procedural steps. This information is highly valuable in efficient assay development.

Isotype controls are used to validate that the primary antibody binding is specific and does not result from background signal due to immunoglobulins binding nonspecifically. Typically, an isotype control is matched to the host species and isotype of the specific primary antibody. For example, IgG2a type antibodies raised in mice can bind strongly to some human leukocytes. Hence, a mouse IgG2a isotype control should be used when analyzing human cells and tissues. Isotype controls are most commonly used in immunoprecipitation, flow cytometry, and immunohistochemistry. In many flow cytometry applications, directly labeled primary antibodies are used. Here, it is important to use the isotype control that is conjugated to the same fluorochrome or label as the primary antibody.