Positive and Negative Controls For Assay Optimisation

With the continual expansion and discovery of new research worldwide in recent years, assay optimization has turned out to be a vital bottleneck. With the recent change from manual to automatic HTS, researchers have seen a wide and significant rise in the number of targets screened. Due to this, there is a need for throughput in the screening environment. The development of robust assays for an automated screening environment remains one of the challenges faced by the pharmaceutical industry. This results from the long period required by the assay optimization process, usually four to twelve months with the use of conventional or local one-factor-at-a-time experiments or, at times, simple two-factor checkerboard experiments.  

Controls For Assay Optimisation

The Importance of ELISA Controls

Appropriate running of controls helps researchers in separating true positive results from likely to fail or false results. However, having these two controls in an experiment will be useful if you need to troubleshoot your protocol. Employing positive and negative control, including ELISA controls in immunoassay, helps confirm a proper and accurate run of experimental activities, especially Luminex assay optimization. 

Positive and negative controls for antibody validation

Positive control in research work should confirm that there is an expression of target antigens on the relevant cells and tissues. On the other hand, the negative control should, however, be made of cells or tissues without a known target protein. While researching, it is best to make use of both controls. Confirm the expression of the needed target module in the sample using a different assay. 

What is positive control? 

This control gives researchers a direct and positive result upon the conclusion of their experimental works. Using a positive test in research works gives "yes" as a result. It is a good indication to determine if researchers have achieved what they aim for in the research or test. Therefore, they are used in the evaluation of a test's validity. There is no exposure of the positive control to the experimental test; it is, however, done parallel to it. A positive control is used in targeting and getting the results researchers expect. It is aimed at a successful test, and upon getting a positive result, the test can be used for an experimental treatment. Should there be a different result expected from positive control, it is best to experiment again (by varying different parameters) until there is a positive result. 

positive control

Applications of positive control

Positive control has several applications in biochemical experiments, including assay optimization. A few applications of positive control includes 

● Diseases detection

● Microorganisms growth observation

● Enzymes presence measurement after doing assay 

What is negative control? 

Just as the name gives an insight, negative control; is an experimental control that is unresponsive to the test. It does not have direct exposure to experiments. It is carried out parallel to the experiment as a control experiment. 

A negative control is used to confirm no response to the reagents or the microorganisms employed in the test, including any other parameters employed during the test. To achieve the desired result with negative control, it is important to make sure that no there is zero net response to the test. Therefore, the negative control is suitable for the identification of influences on the test or experiment. An instance of this effect is that the effect of the contaminant on an experiment can be indicated. 

What differentiates these two controls in assay optimization?

As an experimental control, positive control provides a positive result after experimental exercise, while a negative control does not respond to the test. When it comes to the outcome after experiments or tests, the negative control, just as the name implies, provides a negative outcome while the positive provides a positive result.

While experimenting, the positive control responds to the experiment while the negative does not. A positive control is used in testing experimental validity, while negative control is used in identifying the effect of outside factors on the test. 

What to use as a positive and negative control?

Both controls are applicable in different experiments and tests. While both help achieve different results and responses, it is important to figure out what to use as a control, especially in the case of assay optimization. 

● For the validation of antibodies, cell lines and tissues that endogenously express lack the protein of interest. However, these cells and tissues at greatly used as controls 

● Transfected cells are used as positive controls. (cDNA encoding transfected cells) On the other hand, the negative cells should be made up of only empty vector-transfected cells. 

● Controls with a wide range of protein expression levels 

● Knockout (KO) mice 

● A carefully planned siRNA 

● Xenografts generated from cell lines with a verified level of target expression are a great tool for verifying target specificity. 


Both controls for assay optimization provide a completely experimental response. However, what majorly distinguishes these controls from each other is their response to experiments and tests. Despite their differences, they are both useful in experiments. 

For effective experimental elements, controls are essential to achieve the desired results. They are required and helpful for the maintenance of scientific experiments to remove errors and biases from experiments. For validating statistical analysis, such as optimization and validation of the assay, results of control experiments are useful for experimental analysis. Therefore, experimental reliability can increase by control treatments. 


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