Enzyme-Linked Fluorescent Assay (ELFA)
المؤلف:
APURBA S. SASTRY , SANDHYA BHAT
المصدر:
Essentials Of Medical Microbiology 2021
الجزء والصفحة:
3rd edition , p159-160
2025-08-20
598
It is an modification of ELISA, differs from ELISA in two ways: (i) automated system, all steps are performed by the instrument itself, (ii) Ag-Ab-enzyme complex is detected by fluorometric method. VIDAS and miniVIDAS (bioMérieux) are commercially available systems based on ELFA technology (Fig. 1).
Fig1. miniVIDAs system and reagent strip (first well is solid phase receptacle coated with Ag or Ab and other wells contain various reagents). Source: Department of Microbiology, JIPMeR, Puducherry (with permission).
Procedure: The solid phase receptacle (Fig. 1) present in reagent strip (equivalent to wells in microtiter plate in ELISA) serves as the solid phase; which is either coated with capture antigen (for antibody detection) or antibody (for antigen detection)
- The conjugate used here is either an antigen or antibody labeled with enzyme alkaline phosphatase and the substrate used is 4-methyl-umbelliferyl phosphate
- Following Ag-Ab-enzyme conjugate complex formation; the excess of enzyme conjugate are washed out
- Then the conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-methyl umbelliferone), the fluorescence of which is measured at 450 nm
- The intensity of the fluorescence depends on the concentration of alkaline phosphatase present which in turn depends upon the amount of analytate (target Ag or Ab) in the sample.
Advantages: It has many advantages over ELISA: (i) an automated system, (ii) easy to perform and user friendly, (iii) less contamination chance, (iv) gives quantitative results and (v) more sensitive and specific
Disadvantages: (i) Expensive, (ii) can run only 12–24 number of tests at a time, (iii) can run 2–4 types of tests at a time
Use: It can be used to detect numerous parameters
- Infectious diseases: Markers of hepatitis viruses and HIV (Ag and Ab), Ab to TORCH infection, measles, mumps, varicella, H. pylori and antigen to C. difficile, Rotavirus, etc.
- Other uses: Biomarkers (e.g. procalcitonin), hormones (e.g. thyroid), tumor markers, cardiac markers and screening for allergy.
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