Enzyme multiplied immunoassay technique (EMIT) is a modern antigen-antibody based method normally employed in the determination of the drugs concentration in urine and serum. EMIT has found extensive use in the wake of increased drug abuse and increased need for therapeutic drug monitoring.
Screening for drugs is now a requirement by various institutions in many jurisdictions around for their own administrative reasons. EMIT can be used to tell not only the identity but also the quantity of drugs and other proteins of interest in blood.
Therefore, EMIT straddles between qualitative and quantitative methods of drug monitoring. It can serve any of these two needs and give credible results if performed well.
In this article we shall explain the principle of this method, that is how it works, the general methodology of this technique, interpretation of results, and applications of this method in the modern world.
Principle of Enzyme multiplied immunoassay technique (EMIT)
This immunoassay takes the model of competitive ELISA. The drug/protein in your sample of interest competes for a binding place in the antibody molecule (Commercially procured) with an enzyme-labelled drug or drug metabolite. The epitopes of the two drugs bind to the same antibodies and so there is competition.
The method is pegged on the ability of the antibody to inhibit enzyme activity when unlabeled drug is not available in the sample. The binding of the antibody to the enzyme-labeled drug inactivates the enzyme so it won’t act on its substrate.
However, the free enzyme-labeled drug that is not bound to the antibody will act on its substrate i.e., when the sample contains the drug of interest. Hence, the more the drug there is in your sample, the more the replacement of the enzyme-labelled drug from its complex with the antibody molecule.
That means, there will be more enzyme-labelled drug (free) in the solution than bound to the antibody binding sites. The enzyme which is normally Glucose-6-Phosphate Dehydrogenase (G6PDH) converts glucose-6-phosphate to guconolactone-6-phosphate.
In addition, the enzyme reduces NAD to NADH. The amount of NADH (measured spectrophotometrically) produced is directly proportional to the concentration of the drug of interest in the sample.
Performing EMIT in the Laboratory
To perform this immunoassay, like in many other assays, you need to assemble all the ingredients that you need to use beforehand. These ingredients include the following components:
- Microtiter plate sometimes already coated with the highly specific commercially procured monoclonal antibody that binds to the epitopes of the analyte of interest.
- Enzyme that is linked to a commercially procured analyte that is the same as the suspect analyte of interest in your sample. Most of the time glucoce-6-phosphate dehydrogenase (G6PDH) is used.
- Enzyme-specific substrate that will be acted upon and bring about color change if the analyte of interest is present.
- A test sample that is collected in an appropriate container and handled well to avoid contamination that can lead to false positive results.
To perform the test an appropriate volume (Kit manufacturer’s instructions) of the urine sample is added to the plate, followed by addition of the G6PDH-labeled drug.
The antibody that is specific for both the labeled and the unlabeled drug is added in appropriate volume as directed in the kit insert. The homogenous solution is incubated at the right temperature and period.
The substrate for G6PDH is added and mixture subjected to UV spectrophotometry at 340nm. Since G6PDH reduces NAD to NADH, the amount of NADH measured will be directly proportional to the concentration of the drug suspected in the sample.
Results Interpretation
When there is high amount of NADH as measured spectrophotometrically, then it means that the urine sample contains high amount of the drug of interest.
The drug or drug metabolite was able to competitively replace so much enzyme-drug and that is why so much of the free enzyme-labeled drug was available to react with its substrate.
On the other hand, low amount of NADH if measured spectrophotometrically, then it means that the enzyme-labeled drug was sufficiently bound to the antibody, enzyme inactivated, and so free enzyme-labeled drug was little.
So, there is low concentration of unlabeled (drug of interest) metabolite in the urine sample.
The results for this test must be interpreted with care to avoid misinterpretation that can lead to wrong conclusions. This is because misinterpreted with for example false positive can have dire social and legal consequences.
Therefore, the results must be interpreted in the context of the set cut-off and frequently corroborated with the results from a credible alternative method.
The methods that you can use to ascertain whether such results are accurate or not include mass spectrometry and gas chromatography.
In the US, any immunoassay method that doesn’t conform to the standards set by SAMSHA are not acceptable.
For instance, it has been found that such methods can report those passive inhalers of marijuana as abusers of the same which can lead to unfair verdicts.
Best Applications of Enzyme Multiplied Immunoassay Technique
The best applications of EMIT today include substance abuse monitoring and therapeutic drug monitoring in samples of urine and serum. Let’s now discuss these two applications in detail here.
1. Therapeutic Drug Monitoring
EMIT is used in monitoring the concentration of therapeutic drugs in blood serum. This monitoring is critical particularly you want to know about the delicate balance between therapeutic dose and adverse effects from that drug.
This is true for immunosuppressive drugs and some types of antibiotics where the therapeutic window is quite small.
In such circumstances an excessive dose means problems while a dose that is too small means it won’t work – monitoring using EMIT is key.
For immunosuppressive drugs, there are many reasons why they can be administered. Let’s take an example of case or a transplant rejection reaction and so the drugs are supposed to be given to avert chronic rejection.
If there is too high concentration your immune system will be suppressed to much and make you vulnerable to infections.
If the immunosuppressant is too little, it won’t help to prevent chronic rejection and so you will lose your hard sought transplanted organ. Very sad! isn’t it?
2. Pre-Specified Minimum Concentration Drug Monitoring
There are some drugs whose serum concentration is pre-defined in law and healthcare policies in efforts to prevent substance abuse and safeguard mental wellness. This is the practice in many jurisdictions around the world.
In the US for instance, the minimum and cut-off limits are set by SAMSHA a department of Health and Human services in accordance with the Mandatory Guidelines for Federal Workshop Drug Testing Programs.
Whenever and wherever, there is need to detect suspected substance abuse, the laboratory technologists and scientists employ EMIT as the recommended method that gives credible results that can be used to make important decisions.
EMIT is used to perform urine immunoassays for drugs such as amphetamine, cannabinoids, marijuana, heroin, and morphine.
These drugs have increasing been abused in the recent past and hence the need for these policies and test as deterrent for their abuse.
Conclusion
Enzyme multiplied immunoassay technique (EMIT) is a form of competitive ELISA that is based on antigen-antibody reactions to give results.
EMIT is heavily relied by authorities to determine cases of substance abuse and help in serum therapeutic drug monitoring.
The use of an alternative method like Gas chromatography, and/or mass spectrometry can help in quality control of EMIT especially where the results have serious consequences.
Nice content here
Thanks Mary