Using cell lines for antibody validation - what you need to know to get started

CRISPR-edited cell lines are a useful tool to validate your antibodies before you start an experiment. They ensure you are using high quality reagents, so you can be confident in your results. We explain how you can use our edited cell lines for validation, the challenges to be aware of and how we can help you overcome them.

Antibody Validation using Cell Lines

Using positive and negative controls

Positive and negative controls are an essential part of the antibody validation tool kit. Positive controls give a good indication that the antibody is binding to the target antigen. However, if an antibody is non-specific these results can be misleading. The negative control is used in conjunction to confirm specificity and should consist of tissues or cells where the target protein is known to be absent.

Often it is difficult to find biological material where there is adequate information on the level of protein expressed or if there is a complete lack of the target of interest. However, with the availability of CRISPR gene editing technology, knockout cell lines are fast becoming established as a standard control for antibody validation.

The challenges of antibody validation, and how we can help solve them

Challenge 1: Creating your own knockout cell line. Although CRISPR gene editing technology is relatively simple, the reality of producing clonal cell lines in house can demand significant time and resource. A simple knockout cell line can take upwards of 13 weeks; from the design of reagents, through to selecting for successful clones, and eventually characterizing and validating any resulting cell lines.

Solution 1: Horizon’s ready-made knockout cell lines Our catalog of ready-made CRISPR gene-edited Knockout cell lines has over 2500 gene targets already available for immediate shipping.

Challenge 2: Accessing control material. Finding a biologically relevant material is time consuming. There are a number of considerations, including:

  • is the cell line authentic?
  • is it free from contamination?
  • is it at low passage?
  • does it exhibit the right biology?

Solution 2: Wild-type cell lines. We have characterized the parental wild type cell line used in our human knockout cell line range. For each gene target there is an indication of the expected gene expression, so you can easily decide whether this cell background is relevant to your work.

If you need more guidance on choosing the right cell line for your experiment, we explore these questions further in our blog: 5 tips for choosing the right cell line

Challenge 3: Biological background and genetic variation. Having positive and negative controls in the same biological background is a big bonus as this can make sample handling much more streamlined and results easier to interpret. Traditional sources of positive and negative controls include patient derived materials or cell lines. These usually have high genetic variability, with many non-specific mutations acquired over the years. It is important to know that the desired phenotype is not due to a background mutation.

Solution 3: Characterized models provided with parental cell lines. Our human knockout cell lines come with a parental cell line as standard, enabling you to examine your gene of interest in isolation. Risks that results are due to off-target effects of gene editing (CRISPR) can be mitigated by studying multiple clones, while the parental cell line provides the ideal control ensuring that findings are not due to other experimental variables.

Best Practices

Optimization of experimental parameters for every new antibody is recommended to ensure confidence in your results. Several groups have published papers on the essential considerations to help standardize the quality of the antibody validation process, including the European Monoclonal Antibodies Network and Protein Atlas. You can read our tips on some of the key factors to keep in mind when you optimize and validate any new antibody.

Working with antibody providers

Antibody validation has a key role to play in addressing the current concerns of reproducibility of results in research. There have been many articles highlighting the responsibility of both the individual researcher as well as the antibody provider to make sure antibody validation is undertaken in an application-specific manner.

Many antibody providers, such as Abcam, are turning to knockout cell lines as a means of addressing this issue in the manufacturing process.

Horizon’s knockout Cell Lines

Whether you are an individual researcher looking to validate a single antibody, or an antibody provider with a large catalog, Horizon’s knockout cell lines can help you gain confidence in your valuable research tools.