When using monoclonal antibodies in applications such as preclinical functional animal studies in vivo, the appropriate isotype control antibody is strongly recommended to generate reliable data.

Primary monoclonal antibodies can interact both specifically and non-specifically with Fc receptors on cells, blood proteins, cellular proteins, carbohydrates, lipids and tissues, causing non-specific signals.

The non-specific signal from the isotype control antibody, used at the same concentration or dose as the specific antibody, is used to differentiate a positive signal or result generated by the specific monoclonal antibody.

WHAT IS AN ISOTYPE CONTROL?

An isotype control is an antibody that lacks the ability to bind the target antigen, but matches other properties of the primary antibody. Isotype controls are used as negative controls in place of primary antibodies to determine the contribution of non-specific background staining and to distinguish specific and non-specific antibody staining.

Isotype controls are often used in flow cytometry and immunohistochemistry, where background staining is common. However, they can be used in any assay where the background interferes with the results. They can also be blocking reagents in immunoassays such as Western blots and ELISA.

EXAMPLE: BIO X CELL ISOTYPE CONTROL

BioXCell specializes in in vivo antibody production, so all of their antibodies have a recommended Isotype control. These control antibodies are antibodies of the same species and the same subclass, but they do not react with any molecule present in the body.

For example, the 2A3 monoclonal antibody reacts with trinitrophenol, and since trinitrophenol is not expressed in mammals, it is the ideal antibody to use for an isotype control for rat IgG2a antibodies in in vivo applications.

FUNDAMENTAL REQUIREMENT IN HIGH IMPACT MAGAZINES

The use of appropriate controls, including isotype controls, is imperative for rigorous experimental design and reliable interpretation of data in antibody-based assays. Isotype controls serve as essential negative controls by accounting for non-specific background signal, distinguishing the binding of specific from non-specific antibodies.

While the need depends on factors such as assay type and target sample, isotype controls provide key information about background signal levels. Its inclusion strengthens the specificity of the results by allowing the discernment of a true positive signal. Therefore, it is recommended to incorporate isotype controls to support robust experimental conclusions.

When performing in vivo experiments, isotype controls are extremely useful for analyzing cells and tissues using flow cytometry or microscopy. Tissues contain many proteins to which antibodies can bind nonspecifically. Using an isotype control antibody helps determine the level of background signal in your experiment.

Subsequently, samples labeled with the isotype control can be compared to those labeled with the specific antibody. Any signal greater than the isotype control is likely due to specific binding of the target antibody.

USE CASE: REVERSAL OF IDO-MEDIATED CANCER IMMUNE SUPPRESSION THROUGH SYSTEMIC DEPLETION OF KYNURENINE WITH A THERAPEUTIC ENZYME

Scientists at the University of Austin, in Texas, USA, published in 2018 a study based on how tryptophan catabolism in the tumor microenvironment can mediate immune suppression through the upregulation of the IFNg-inducible indoleamine 2,3-dioxygenase (IDO1). and/or ectopic expression of the enzyme tryptophan 2,3-dioxygenase (TDO), commonly limited to the liver.

Whether these effects were due to the shortage of tryptophan in the tumor microenvironment or whether they were mediated by the presence of kynurenine due to the effect of enzymatic action was unclear.

With this study they aimed to demonstrate that the administration of a pharmacologically optimized enzyme that degrades kynurenine into immunologically inert metabolites would inhibit tumor growth, resolving the controversy between the two possible explanations.

Being an incredibly delicate metabolic environment, the need to exclude any possible background noise was twofold: Not only would it lead to clearer and more decisive results but it would allow the study to be published in one of the highest impact journals in the field, whose requirements publications require these kinds of controls.

In this case, they administered the LTF-2 clone, produced by Bio X Cell, to perform this isotype control.

Request a quote for isotype controls at Abyntek Biopharma

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