EMIT - ENZYME-MULTIPLIED IMMUNOASSAY TECHNIQUE

E- Enzyme

M- Multiplied

I- Immunoassay

T- Technique

04/08/2023Chemical Pathology III

2

INTRODUCTION

Emit is: Cheapest and simplest technique An analytical method used in clinical toxicology Commonly used in screening blood (serum) for

TDM (therapeutic drug monitoring) and as a primary screening procedure of abused drugs and their metabolites in the urine.

It is the first homogenous assay to be widely used. The separation using antibody specificity (antigen

binding).

04/08/2023Chemical Pathology III

3

Measurement of enzyme-substrate reactions using visible spectroscopy, and standard curve.

Produces reliable results The test utilizes antibodies that are enzyme linked

and react only with the specific substance in the sample (urine or blood) is positive with the particular drug being tested.

EMIT (Enzyme Multiplied Immunoassay Technique) is the screening technology used for urine drug testing. GC/MS (Gas Chromatograph/Mass Spectrometry) is considered a confirmatory testing technique.

04/08/2023Chemical Pathology III

4

COMPONENTS OF THE ASSAY:

Drug Antibody Substrate Enzyme bound to drug

04/08/2023Chemical Pathology III

5

PRINCIPLE OF EMIT

Enzyme multiplied immunoassays (EMIT) have a wide application in therapeutic and illicit drug monitoring. In this type of assay, a sample of interest with the analyte (drug) is added to a fixed quantity of enzyme-bound drug, and the anti-drug antibody. After the addition of substrate, absorbance measurements are taken at time intervals to determine the speed of the enzyme reaction.  

04/08/2023Chemical Pathology III

6

The more free drug in the sample, the faster the enzyme reaction because only the unbound enzyme-drug complexes are capable of binding the substrate. The method can be used for whole blood, serum, or urine.

  

04/08/2023Chemical Pathology III

7

In other words Ag being measured competes for the Ab binding sites with the antigen that has been labeled with an enzyme ---->the antibody reagent blocks any enzymatic activity when it is bound with the reagent enzyme Ag complex (thus preventing the formation of the product with a substrate) ----> then the free Ag-enzyme complexes compete with the Ag (in the sample) ----> forming a color change which are proportional to the concentration of the Ag present in the tested sample.

 

04/08/2023Chemical Pathology III

8

04/08/2023Chemical Pathology III

9

PROCEDURE OF EMIT

Mix sample containing drug with antibody Incubate Add fixed quantity of enzyme bound drug Incubate Add substrate and cofactor Incubate Measure absorbance 15-45 seconds after substrate

addition Absorbance > Reaction rate > Drug concentration Non linear relationship between Absorbance and

Concentration

04/08/2023Chemical Pathology III

10

CLINICAL UTILITY

Enzyme multiplied immunoassay technique is used for Drugs, hormone, and metabolite determination.

  The clinical value and sensitivity of serum

caffeine clearance measurement has been evaluated as an indicator of hepatic disease. After a 17 hour caffeine exclusion period, 300 mg of caffeine citrate was administered orally to the study subjects.

04/08/2023Chemical Pathology III

11

EMIT is widely used in therapeutic and

illicit drug monitoring (e.g: in Determination of Serum Digoxin, Cocaine, and Marijuana etc.)

   

04/08/2023Chemical Pathology III

12

SENSITIVITY OF EMIT

Results for abused drugs in urine, as obtained by radioimmunoassay (RIA), the "Enzyme Multiplied Immunoassay Technique" ("EMIT"), and hemagglutination inhibition (HI), were compared with each other, with fluorimetry, and with thin-layer chromatography (TLC).

04/08/2023Chemical Pathology III

13

The immunoassays and fluorimetric methods were highly sensitive (30 µg/liter to 2 mg/ liter). (New York State Drug Abuse Control Commission, Testing and Research Laboratory, 2003).

This method is less sensitive when compared to Elisa technique.

04/08/2023Chemical Pathology III

14

ADVANTAGES OF EMIT

Homogeneous means there is no need of washing excess of labelled enzymes.

Long shelf-life Assays are well established More specialty assays are available It is a competitive assay Reagent separation is not required Is faster Smoller molecules are measured

04/08/2023Chemical Pathology III

15

DISADVANTAGES OF EMIT

Many interferences Less sensitive compared to Elisa in

hormone determination False negatives are common Tolmetin metabolites Aspirin metabolites

04/08/2023Chemical Pathology III

16

THANK YOU!

04/08/2023Chemical Pathology III

17

REFERENCES

1. J. E. McDonagh, V. V. Nathan, I. C. Bonavia, G. R. Moyle, and A. R. Tanner(1991), Caffeine clearance by enzyme multiplied immunoassay technique: a simple, inexpensive, and useful indicator of liver function. Vol32,No 6,page 681–684.

2. Jill Yeakel, the center for foreinsic science research and education: Immunoassay Techniques (PDF).

3. Marja E. Koivunen, Richard L. Krogsrud (2006), Principles of Immunochemical Techniques Used in Clinical Laboratories review (PDF), Antibodies Incorporated, Davis, CA.

4. New York State Drug Abuse Control Commission, Testing and Research Laboratory, Brooklyn, N. Y. 11217.