What is difference between control and calibrator?

In order to continue enjoying our site, we ask that you confirm your identity as a human. Thank you very much for your cooperation.

Standards, calibrators, and controls: if you’re reading this, you probably know that they are all essential to immunoassay development, manufacturing, and quality control. At times, some of these terms are used interchangeably to refer to the same thing (or nearly the same thing).

That’s a mistake. In truth, standards, calibrators, and controls are all quite different—and knowing the difference could make or break your next immunoassay project.

Knowing the Difference Can Make or Break Your Project

First, a quick primer: an immunoassay involves chemical reactions between clinical samples and reagents that are performed under standardized conditions. The resulting response is related to the concentration of analyte in the sample. Regardless of the form the immunoassay takes, the relationship between response and concentration needs to be estimated (i.e., “calibrated”).

What are Standards in the Context of Immunoassays?

A standard is a material that contains the same analyte as the intended target analyte and is in the same matrix. Standards represent an ideal that is very difficult—if not impossible—to attain in a high-volume commercial manufacturing environment. An example of a standard is the drug digoxin dissolved in an authentic human serum matrix.

What are Calibrators in the Context of Immunoassays?

Often, what are called standards in an immunoassay method are actually calibrators: either they are not in the same matrix as the target analyte (as is a standard), or else an analytically pure and immunologically active analyte is not available.

Digoxin standards/calibrators provide a case-in-point. The matrix in which they are prepared closely mimics normal human serum, but it is prepared from recovered Acid Citrate Dextrose (ACD) plasma by clotting the pooled plasma with the addition of calcium chloride and removing the clots by filtration. The resultant material is only about 60 mg/mL protein rather than the 70 mg/mL protein of authentic normal human serum due to the dilution of the plasma by the anticoagulant ACD solution used during the blood collection. Preservatives such as sodium azide are also added to prevent bacterial degradation during storage. This is not a substance anyone would want in their circulatory system, but it does the job of providing reference points in a digoxin immunoassay.

(If you’re already thinking this is a complicated topic, you’re not wrong: the scientists at DCN Dx have spent decades becoming experts in this field. Fortunately, DCN Dx offers courses that provide startups, students, and faculty who are starting development programs with the fundamental knowledge and skills they need for successful lateral flow projects. However, if you’ve gotten to this point and you’re hungry for more, read on—we dive a lot deeper into this topic below.)

Sometimes this synthetic human serum must be further processed to make the desired calibrators. Thyroxine (T4) calibrators are prepared in charcoal-stripped defibrinated plasma because the pooled plasma contains normal amounts of T4. To make calibrators in the hypothyroid range requires a starting material that contains little or no T4. Charcoal stripping removes protein, so the resultant material is usually about 50 mg/mL protein. For total T4 calibrators, this lower protein level does not matter. However, for free T4 calibrators where the analyte concentration is a cross-product of the analyte level and the endogenous binding protein level, the protein concentration must be increased by ultrafiltration to remove excess water.

Charcoal stripping works well for small molecules such as drugs and some hormones, but protein markers such as human chorionic gonadotrophin (hCG) or thyroid-stimulating hormone (TSH) cannot be removed by charcoal stripping. Normal plasma contains typical amounts of TSH and some hCG. Again, the creation of calibrators in the lower ranges requires starting with a matrix that has no or very little analyte.

Immunoaffinity stripping using antibodies specific for the analyte has been demonstrated on a research scale, but it is impractical for high-volume commercial use. One solution that has been used in commercial practice is to use animal serum. These matrices may contain the animal version of the hormone in question (e.g., equine TSH), but antibodies raised against the human version of the hormone do not recognize the animal hormone.

Sourcing the analytes for preparation of calibrators can also be challenging. Drugs and small hormones are often commercially available in pure form and can be quantitated by weight or UV/Vis measurement. Protein hormones are much more problematic. Originally, analytes such as hCG and TSH were isolated from human tissues obtained from cadavers. This approach works, but it presents supply and biohazard issues—human tissues can (and do) contain human pathogens.

The advent of genetic engineering has provided an excellent solution to this supply problem— human hormones can be made in a mammalian cell culture. Attempts to make human hormones at a lower cost in bacterial cell cultures have failed. This is because mammalian hormones are glycosylated and the presence of these sugars changes both their biological function and their recognition by antibodies raised to recognize authentic human hormones.

Quantitating protein hormones so that one can make defined calibrators is a challenge. UV/Vis measurements that work well for drugs and small molecule hormones do not suffice for larger protein molecules. Pure proteins tend to aggregate, and aggregated proteins do not quantitate reproducibly in immunoassays. Adding irrelevant proteins such as albumins can reduce aggregation, but these proteins have UV/Vis absorbances as well and are often added in huge excess over the concentrations of the target analytes.

The common practice for calibrating these analytes is to use international reference preparations (IRP). These materials are prepared and assigned values by organizations such as the World Health Organization (WHO). They are prepared in large batches and lyophilized for long-term storage. The user reconstitutes the material and spikes it into their own matrix and then value assigns their own calibrators based on that international reference preparation.

Because the original preparations were calibrated in bioassays, and given the difficulties cited above for precise quantitation of protein hormones, the calibration given by these IRPs was frequently in international units (IU) or derivatives of IUs such as milliIU or even microIUs. These units bear no relationship to moles or exact weights of analytes, but the package insert that comes with the IRP will often provide an approximate conversion of IU to micrograms. Nonetheless, these IRPs do help achieve some degree of consistency between different methods.

Despite attempts to build large lots of IRPs, the stocks of these materials inevitably run out, prompting serial IRPs that all differ slightly from one another, further complicating the analysis.

What Are Controls in the Context of Immunoassays?

Controls used in immunoassays are a very different material. Their sole purpose is to measure whether a given method is providing the same results day after day and month after month. They may, in fact, be of the same composition as the calibrators supplied by the manufacturer. However, they are not used to calibrate the assay—only to check the assay’s consistency over time.

Commercial controls that contain a bewildering array of analytes in the same vial are also used, and they provide an important independent reference point for a commercial assay. Calibrators and controls made in the same manner can drift over time in the same way, and this drift can go undetected. An independent control is best when it is available.

Controls also provide a window into the different ways various methods see the same analytes. Controls usually come with a very long list of values for a given analyte, depending on which method is used for the analysis. This does not mean that some manufacturers are correct and others are not; it simply means that different methods using different antibodies and formats report different results.

To be useful in commercial practice, reagents need to have a reasonable shelf life under defined conditions. Eighteen months is a typical goal as longer shelf lives generate unsold materials that are costly to prepare and store. Liquid ready-to-use calibrators and controls are the easiest to use and provide the least opportunity for user error. Lyophilization is used when the material is not stable enough to ship in a liquid form. Reproducible lyophilization is an art form that takes careful formulation and meticulous attention to the manufacturing process.

Packaging can play a major role in the performance of calibrator and controls. Some analytes must be packaged in brown glass containers because they are light sensitive. Others require antioxidants to maintain activity. Even the stopper in the vial can have a critical influence: slight changes in the polymer formulation of the stopper can change a stopper from being an inert part of the packaging to an absorber of the analyte in question.

Serum and plasma are not the only matrices that are tested in immunoassay. Other clinical matrices include urine, cerebrospinal fluid, amniotic fluid and saliva. Each presents their own analytical, base material supply, production, and stability issues that must be addressed by the assay development team as they craft an assay for real world use.

In summary:

• Standards are the ideal method for evaluating assay performance but are seldom a realistic option
• Calibrators are a pragmatic solution that mimic a standard and allow reference points in comparing assay results to analyte levels in patient specimens
• Controls provide a means of evaluating an assay’s consistency, or reproducibility, in providing accurate analytical results from a patient specimen

Need to get a head start on your organization’s assay development? Looking to quickly overcome a frustrating development issue? DCN Dx’s customized hands-on lateral flow training courses are the answer. These private courses are customized to your specific needs and delivered in DCN Dx’s lab using your own assay. Perfect for startups and university research groups in the beginning stages of lateral flow development projects; manufacturing groups; and research groups in mid- to large-size organizations.

For more information, click here.