HIV ELISA: Introduction, Principle, Procedure, Result Interpretation and Clinical Significance

HIV ELISA test result

HIV ELISA Introduction 

HIV ELISA result as shown above image. In HIV testing, a blood or saliva specimen is collected for testing typically by the use of indirect ELISA-based tests. It is a screening tool for HIV detection, but not diagnostic. Diagnosis requires further testing by Western blot due to potential false positives. ELISA stands for Enzyme-Linked Immunosorbent Assay. It comes under antigen and antibody reaction test and is useful for identification of antigen or antibody of following specimens serum, urine, CSF, sputum, semen, supernatant of culture, stool, etc. It is also applicable for qualitative as well as quantitative determination of antigen or antibody. In qualitative test determines antigen or antibody is present or absent whereas in quantitative determines the quantity of the antibody in titer and the titer is the highest dilution of the specimen usually serum which gives a positive reaction in the test.

There are four major types of ELISA

  1. Direct ELISA: Antigen-coated plate and uses for screening antibody.
  2. Indirect ELISA: Antigen-coated plate and uses for screening antigen/antibody.
  3. Sandwich ELISA: Antibody-coated plate and screening antigen)
  4. Competitive ELISA (screening antibody)

Principle of HIV ELISA (Indirect ELISA) 

In this technique, the microtitre wells are coated with an antigen. It is then reacted with antibody first( specimen) and then secondary antibody ( anti-human globulins) is added, which is already tagged with enzyme and leads to the color product after addition of specific substrate. After few minutes ( time according to manufacturers) stop solution is added and it is then measured by ELISA reader. The intensity of the color is directly proportional to the concentration of antibodies present in the specimen.

Cut off Value

Cut-off value: provided in the kits by the manufacturer. The cut-off value defines a range in which 90% of the normal population is negative below the cut-off value and 10% of the normal population is positive above the cut-off value. ELISA is a semi-quantitative method. The calculation is done as follows. The units of ELISA is  optical density (OD)  ratio:
Sample value= sample OD/cut-off OD

Requirements for HIV ELISA

 

  1. Solid-phase support:- 96-well microtiter well or polystyrene beads or tubes. Microtitre is well-round, flat, or C-shaped and it is coating with antigen or antibody. Antibodies or antigens are absorbed onto plastic surfaces in an alkaline buffer(carbonate or bicarbonate, PH -9.0) 37 ⁰C for 1 to 2 hours at room temperature overnight. Wash off unbound reagent. Add blocking solution (1% sodium casein or gelatin or BSA) for 30-60 minutes at 37⁰C.  Rewash and then sugar solution (1% glucose, sucrose, mannose, or maltose) for 30 minutes at room temperature. Plates are then dried rapidly using steam of nitrogen or a vacuum and finally tore at 4⁰C.
  2. Washing solution:- Phosphate buffer saline (PBS) containing 0.05% Tween 20 ( PBS/T).
  3.  Diluent buffer:-Phosphate buffer saline (PBS) containing 0.05% Tween 20.
  4. Enzyme –substrate system:- It consists of an enzyme-linked to a specific antibody or antigen and a specific substrate containing chromogens. Stop solution, Enzyme -substrate Initially the substrate should be colorless. After degradation by the enzyme, it should be strongly colored. Enzymes and their respective substrate, chromogen and stop solution are as follows for  Alkaline Phosphatase (enzyme), para-Nitrophenylphosphate (pNPP) (substrate),  para-Nitrophenylphosphate + diethandamine+ magnesium chloride (MgC2)( chromogen), 1 M NaOH (stop solution), Horseradish Peroxidase (H2O2 ), Tetramethylbenzidine + Phosphate – Citrate buffer, 1 M and H2SO4, Horseradish Peroxidase, H2O2, O – Phenylenediamine + HCl, 1 M HCl.
  5. Stop solution
  6. ELISA Reader
  7. Incubator
  8. ELISA washer ( optional)
  9. Distilled water
  10. ELISA washer wash solution
  11. Measuring cylinder
  12. Adhesive tape ( provided in most kits)
  13. ELISA log sheet
  14. Controls ( Negative control and positive control)

Test procedure of HIV ELISA

  1. Load samples in microtiter plate wells.
  2. Incubate
  3. Wash the plate
  4. Add diluent conjugate
  5. Incubate
  6. Wash the plate
  7. Add diluted TMB substrate
  8. Incubate
  9. Add stop solution
  10. Take reading using a spectrophotometer as shown below-

Result Interpretation of HIV ELISA

ELISA is applicable to measure analytes both qualitatively as well as quantitatively.

Reactive test result:  Value of test is equal or greater than Cut off Value

Non-reactive test result: Value of test is less than Cut off Value

Negative control: Non-reactive test result

Positive control: Reactive test result

Clinical Significance of ELISA

ELISA can be used for many purposes as shown below-

  1.  Rapid antibody screening tests for Human immunodeficiency virus (HIV)
  2. Detection of other viruses.
  3. Detection of bacteria.
  4. Detection of fungi.
  5. Detection of autoimmune diseases markers.
  6. Detection of food allergens.
  7. It uses in blood typing.
  8. Detection of the presence of the pregnancy hormone hCG.
  9. It is also an application to the laboratory, clinical research, and even forensic toxicology.

Interfering Factors of ELISA Test Results

Following factors which affect the ELISA result are as follow as-

  1. Plate Assay: the shape and quality of the wells, the material of the plate, potential pre-activation, even or uneven coating
  2. Buffer: pH, contamination
  3. Capture and detection antibody: incubation time, temperature, specificity, titer, affinity
  4. Blocking Buffer: cross-reactivity, concentration, contamination
  5. Target antigen: conformation, stability, epitopes
  6. Enzyme conjugate: type, concentration, function, cross-reactivity
  7. Washes: contamination, frequency, volume, duration, composition
  8. Substrate: quality/manufacturer
  9. Detection: instrument dependent factors
  10. The reader or human error

Advantages of ELISA

  • Specific and  Sensitive, wide application
  • Equipment cheap and  easily available
  • Reagents “Cheap”, long shelf life
  • Assays may be rapid
  • Simultaneous assay, variety of labels
  • Potential for automation
  • No radiation hazards

 Disadvantages of ELISA

  • Contamination
  • Expertise required to label and purify conjugates
  • Susceptible to interference from non-specific factors

References

  1. https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay
  2. https://www.who.int/diagnostics_laboratory/faq/elisa/en/
  3. https://www.ncbi.nlm.nih.gov/books/NBK555922/
  4. http://www.biobest.co.uk/diagnostics/techniques/elisa-how-does-the-test-work.html
  5. https://stanfordhealthcare.org/medical-conditions/sexual-and-reproductive-health/hiv-aids/diagnosis/elisa.html
  6. https://www.sciencedirect.com/topics/immunology-and-microbiology/elisa
  7. Textbook of Medical Laboratory Technology by Praful B. Godkar, Darshan P. Godkar
  8. Textbook of Medical Laboratory Technology by Ramnik Sood (2006)
  9. https://www.ncbi.nlm.nih.gov/books/NBK555922/
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