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HBsAg ELISA Introduction

HBsAg ELISA result as shown above picture and it stands for hepatitis B virus surface antigen. A viral disease, chronic hepatitis B is an important health problem that affects over 350 million people worldwide. The HBsAg has been shown to be an early marker of infection by the hepatitis B virus and therefore, qualitative HBsAg assay, enzyme immunoassays (EIAs) have been used as screening tools for several decades. But nowadays, quantification of HBsAg is in the spotlight as a new potential marker for monitoring on-treatment response as several studies have reported a good correlation between levels of serum HBsAg and covalently closed circular DNA (cccDNA) before and after antiviral therapy. Serum antigen, HBsAg levels reflect the concentration of intrahepatic hepatitis B virus cccDNA and may ultimately prove valuable in the management of patients with chronic hepatitis B.

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 uses for screening antigen.
4. Competitive ELISA: It uses for screening antibodies.

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 (MgCl₂ )( 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 HBsAg ELISA

1. Add sample in well plus anti HBSperoxidase solution
   Incubate the at 37°C.
2. Wash the plate.
3. Add diluted TMB substrate.
4. Incubate at room temperature.
5. Add stop solution ( sulphuric acid).
6. Read the rest using an ELISA reader or spectrophotometer as shown below-

Result Interpretation of HBsAg 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 hepatitis B virus.
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
• The benefits of Competitive ELISA are that there is less sample purification needed, it can measure a large range of antigens in a given sample, can be used for small antigens, and has low variability.

 Disadvantages of ELISA

• Contamination
• Expertise required to label and purify conjugates
• Susceptible to interference from non-specific factors
• Direct ELISA disadvantages include its low sensitivity when compared to the other types of ELISA and its high cost of reaction.
• The only major disadvantage of indirect ELISA is the risk of cross-reactivity between the secondary detection antibodies.
• The sandwich ELISA has the highest sensitivity among all the ELISA types but the major disadvantages of this type of ELISA are the time and expense and the necessary use of “matched pair” like divalent/ multivalent antigen and secondary antibodies.
• The competitive ELISA has a low specificity and can not be used in dilute samples.

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/