What is ELISA? (Enzyme-linked Immunosorbent Assay)

Enzyme-linked Immunosorbent Assay (shortened as ELISA) is used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and color changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration.

Development and brief history

Before the advent of ELISA, radioimmunoassay employing radioactively-labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.

In 1960s, Enzyme linking process was developed by two different teams spearheaded by Stratis Avrameas and G. B. Pierce. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath. Independent research papers published in 1971 by Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands produced the knowledge that go into making ELISA.

The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity)

Technically, newer assays use reporters that are not enzymes in most cases, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.

ELISA Principle

ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.

Types of ELISA

Direct ELISA

Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Direct Elisa

Simple Procedure
  1. The antigen is diluted to a final concentration of 20 μg/ml in PBS. The wells of a PVC microtiter plate are coated with the antigen of 50 μl in the top wells of the plate.
  2. The plate is covered with adhesive plastic and incubated for 2 hours at room temperature.
  3. The coating solution is discarded and completely removed by flicking the plate over a sink and the remaining drops are removed by patting the plate on a paper towel.
  4. The remaining protein-binding sites in the coated wells are blocked by adding 200 μl blocking buffer.
  5. The plate is covered with an adhesive plastic and incubated for at least 2 hours at room temperature.
  6. The plate is washed twice with PBS.
  7. 100 μl of the antibody is added to the blocking buffer before use.
  8. The plate is covered with an adhesive plastic and incubated for 2 hours.
  9. The plate is washed four times with PBS.
  10. the substrate solution is dispensed in each well.
  11. Post color development, stop solution is added to the wells.
  12. Absorbance of each well is studied against a plate reader.

Indirect ELISA

The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.

Indirect Elisa

 Simple Procedure
  1. The antigen is diluted to a concentration of 20 µg/ml in PBS. The wells of a PVC microtiter plate are coated with the antigen by pipetting 50 µl of the antigen dilution in the top wells of the plate.
  2. 200 µl blocking buffer is added to block the remaining protein-binding sites in the coated wells with 5% non-fat dry milk.
  3. The plate is covered with adhesive plastic and incubated for at least 2 hr at room temperature.
  4. The plate is washed twice with PBS.
  5. 100 µl of diluted primary antibody is added to each well.
  6. The plate is covered with an adhesive plastic and incubated for 2 hours at room temperature.
  7. The plates should be washed with PBS four times
  8. 100 µl of conjugated secondary antibody diluted at optimal concentration in blocking buffer is added.
  9. The plate is covered with adhesive plastic and incubated for 1 – 2 hours at room temperature.
  10. Again, the plates are washed with PBS four times.

Sandwich ELISA

The assay is highly efficient in sample antigen detection and quantification. The assay is suitable for antigens that contain at least two antigenic epitopes capable of binding to antibody. Both monoclonal and polyclonal antibodies are widely used as capturing and detection antibodies in Sandwich ELISA systems. The quantification of antigens happens between two layers of antibodies and therefore the method is termed as sandwich ELISA.

The assay is more sensitive and specific than direct or Indirect ELISA i.e. up to 2 to 5 times. The difficulty in sandwich ELISA lies in optimization and selecting tested pair antibodies. This ensures antibodies chosen are detecting different epitopes on the target protein without interfering with other antibody binding. As a high precision assay, the sample need not be purified before analysis.

 

  Simple Procedure

  1. The wells of the microtiter plates are coated with the capture antibody at 1–10 μg/mL concentration in carbonate/bicarbonate buffer. In case unpurified antibodies are used, the concentration of the protein sample should be 10 μg/mL) to compensate for the lower concentration of specific antibody.
  2. The plate is covered with adhesive plastic and incubated at 4°C for 12 hours.
  3. The coating solution is discarded and the plate is washed twice by 200 μL PBS. The PBS is also removed completely by flicking the plate over a sink and the by patting the plate on a paper towel.
  4. The remaining protein binding sites in the wells are blocked by 200 μL blocking buffer (5% non-fat dry milk/PBS) per well.
  5. The plate is covered with the adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4°C.
  6. The plate is washed twice with 200 µL PBS.
  7. The diluted samples of 100 μL is added to each well. The signals exhibited are compared with a standard curve. Both standards and blank with each plate are incubated for 90 minutes at room temperature.
  8. The concentration range is optimized to obtain a standard curve. This ensures the standards spans the detection range of antibody binding.
  9. the samples are removed and the plate is washed twice with 200 μL PBS.
  10. 100 μL of diluted detection antibody is added to each well. It is important to ensure detection antibody recognizes a different epitope on the target protein to the capture antibody to prevent interference with antibody binding.
  11. The plate should be covered with adhesive plastic and incubated for 2 hours at room temperature.
  12. The plates are washed with PBS four times and 100 μL of conjugated secondary antibody diluted in blocking buffer is added.
  13. The plate is covered with adhesive plastic and incubated for an hour at room temperature.
  14. The plate is washed four times with PBS.
  15. Detection involves enzymes such as Horse radish peroxidase (HRP) and alkaline phosphatase (ALP). Blocking treatment with levamisole or 0.3% H2O2 in methanol. This helps in hindering nonspecific signal.

Competitive ELISA

The assay is the most precise of all ELISA types and the assay is based on competitive binding.

 

Competitive ELISA
 Simple Procedure
  1. Unlabeled antibody is incubated with the sample antigen.
  2. The incubated complex is added to the 96-well plates coated with the same antigen in the complex
  3. The unbound antibody is washed away from the plate.
  4. More antigens in the sample leads to lesser antibodies binding to the antigens in the well, hence the procedure is termed competitive.
  5. Secondary antibody conjugated with the enzyme is added to the well.
  6. A suitable substrate is added and enzymes elicit a chromogenic or fluorescent response.
Selection and Coating
  1. Optimizing plate condition for antigen or capture antibody is of paramount importance in developing assay for a specific antigen.
  2. Microplate with minimum protein binding capacity of 400 ng/cm² are usually chosen for running ELISA. The coefficient of variation of protein binding should be less than 5%.
  3. The plate colors are largely determined based on signal detection.
  4. Optimizing plate condition for antigen or capture antibody is of paramount importance in developing assay for a specific antigen.
  5. Usually, polystyrene bottom plates and opaque plates are used for signal detection. While Polystyrene plates are used for colorimetric signals, opaque plates are used for fluorescent and chemiluminescent signals.
  6. It is important to inspect the plates before use as scratches and imperfections would lead to deviations in the data obtained from the assay.
  7. The coating of the plate is achieved by adsorption of the protein to the plastic of microplate i.e. through Hydrophobic interactions between the plastic and non-polar protein.
  8. The plates are coated by adding 2–10 μg/mL solution of protein dissolved in an alkaline buffer. After incubating the plates for at least 12 hours at less than 37° after removing coating solution, blocking buffer is added to ensure binding surfaces of the plastic well are covered.

Buffers

Buffers help in adsorbing antibodies and antigens that are not binding to the surfaces of the coated plate. Largely, the sensitivity of the assay depends on the blocking buffer as it reduces background signal and enhances the signal to noise ratio. An ideal buffer is an agent that eliminates background completely without impacting the epitope for antibody binding.

Detection Strategies

As a biochemical technique, ELISA is mainly used to detect the presence of an antigen or antibody in a sample. Therefore, detection occupies an important role in ELISA. In simple terms, the final step in ELISA always involves adding a signaling agent to the enzyme to produce a visible signal i.e. color change.

Based on detection strategies, ELISAs are classified into three groups: Chromogenic assays, Chemifluorescent assays and chemiluminescent assays.

Some enzymes on addition to the substrate produces a reaction that fluoresces when light particles are emitted at a specific wavelength. When these enzymes find utility in ELISAs, those assays are called Fluorescent immunoassays. Fluorescence units detected are directly proportional to the quantity of analyte in sample.

Chromogenic assays result in colored reaction product that absorbs in light in the visible spectrum and therefore visible to naked eyes. The antigen-antibody complex reacts with the substrate of choice giving rise to colored reaction product. Like Chemifluorescent assays, the color changes are directly proportional to the amount of the analyte.

 

 

ELISA FAQ

Can someone simplify Enzyme Linked Immunosorbent Assay (EIA)?

Enzyme-linked Immunosorbent Assay (shortened as ELISA) is used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and color changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration.

Before the advent of ELISA, radioimmunoassay employing radioactively-labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.

In 1960s, Enzyme linking process was developed by two different teams spearheaded by Stratis Avrameas and G. B. Pierce. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath. Independent research papers published in 1971 by Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands produced the knowledge that go into making ELISA.

The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity)

Technically, newer assays use reporters that are not enzymes in most cases, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.

Principle

ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.

To know more about ELISA Types, advantages and disadvantages and methods, refer ELISA page in Mybiosource Learning Center.

What are the advantages and limitations of using Western-blot vs. ELISA vs. flow cytometry?

First, let us discuss the definition of Western blotting, ELISA and Flow cytometry in simple language. You can always follow the link to get more detailed information on these assays/methods.

ELISA or Enzyme-linked Immunosorbent Assay is useful in identifying a substance using antibodies and color changes. 

Western blotting is used to separate and identify proteins. Through gel electrophoresis, the proteins are separated based on molecular weight. Find protocol and detailed explanation on western blotting in mybiosource learning Center.

Flow cytometry is useful in analyzing physical and chemical properties of particles. In biological research, cell components are fluorescently labelled and then excited by a laser beam to emit light at different wavelengths. Relative granularity, size, fluorescence intensity and internal complexity is mainly studied via flow cytometry. Find protocol and detailed explanation on flow cytometry in mybiosource learning Center.

Advantages and Limitations of ELISA, Western Blotting and Flow Cytometry

  1. The sensitivity and specificity of ELISA is 98%.
  2. The sensitivity and specificity of Western blot/Flow cytometry is 100%.
  3. Running ELISA is cheaper compared with western blot and Flow Cytometry.
  4. Western blot is time consuming due to lengthy and tedious procedures i.e. 6-16 hours.
  5. Significant amount of time is spent on processing the western blot for detection.
  6. Quality of antibody/antigen is important to both ELISA and Western blotting.
  7. ELISA Kits are readily available for well-known target proteins/substances.
  8. Factors affecting western blot results include transfer method, type of membrane used, blocking buffer, reagents and probing techniques.
  9. Flow cytometry requires management by a highly trained specialist and continuous maintenance by service engineers as complex instruments are prone to microfluidics system blockages in addition to cleaning and laser calibration.
  10. Advantages of flow cytometry include high speed analysis depending on the flow rate, measures both single and large number of cells, measures multiple parameters, quantifies fluorescence intensities, sorts predefined population of cells, in addition to identifying small populations.

How do PCR and ELISA differ in their disease diagnostic abilities?

From HIV detection point of view, the approved window period for ELISA test is 90 days or 3 months i.e. the maximum amount of time taken for human body to produce antibodies.

The PCR test can be used for HIV diagnosis even within 72 hours. Polymerase chain reaction (PCR) tests are used to detect HIV’s genetic material, called RNA. The test is not widely used as it is expensive compared to both western blot and ELISA.

ELISA and Western blot are known for sensitivity and specificity for antibodies/antigens. On the other hand, Polymerase Chain Reaction (PCR) is used to amplify DNA and RNA through reverse transcription.

The Polymerase chain reaction (PCR), one of the most powerful technologies in molecular biology, helps in amplifying or copying specific sequence of DNA or complementary template employing oligonucleotides, heat-stable DNA polymerase and thermal cycling. With each cycle, the number of target molecules is doubled theoretically, and DNA is exponentially amplified. Find protocol and detailed explanation on PCR in mybiosource learning Center.

ELISA, in contrast, used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and colour changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration. 

How do direct and indirect ELISA differ?

Direct ELISA: Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Indirect ELISA: The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.

What is best between sandwich elisa and competitive elisa as their advantages are almost same?

It is basically horse for the courses logic!

Sandwich ELISA is mainly meant for detecting antigens with two antigenic epitopes capable of binding to antibody.

Both monoclonal and polyclonal antibodies are widely used as capturing and detection antibodies in Sandwich ELISA systems. The quantification of antigens happens between two layers of antibodies and therefore the method is termed as sandwich ELISA.

The assay is more sensitive and specific than direct or Indirect ELISA i.e. up to 2 to 5 times. The difficulty in sandwich ELISA lies in optimization and selecting tested pair antibodies. This ensures antibodies chosen are detecting different epitopes on the target protein without interfering with other antibody binding. As a high precision assay, the sample need not be purified before analysis.

The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output.

Conclusion: Sandwich ELISA for detection vs. Competitive ELISA for measuring concentration.

How are antibodies detected?

 Initially, identification and characterization of antibodies were limited to the field of scientific research. As the measurement and identification of bacterial infections, viral infections, autoimmune diseases and allergies became more and more important in clinical settings, methods of detecting antibodies have evolved big time and newer/efficient methods are being offered.

Two important methods for detection of antibodies actively used are immunoprecipitation assay and immunoblotting. While there are assays that are superior to immunoblotting, the cost factor (specific to clinical settings) plays a role in making those assays economically not viable.

Immunoprecipitation

To analyze a protein, you must have a reliable detection system that unambiguously enables you to follow the target protein. This is especially true when the target molecule is in crude or even semipurified form. The purification of a bioactive molecule is frequently accomplished by using a definitive assay designed to recognize a property of the target protein. You can read and find protocol of Immunoprecipitation in mybiosource learning center.

Immunoblotting

The techniques use antibodies to identify target proteins from sample protein species. It involves identification of target protein via antigen-antibody specificity.  Proteins are separated by electrophoresis and transferred onto membranes. The membrane is overlaid with a specific target (primary antibody) and a secondary labeled antibody i.e. enzymes or radioisotopes. You can read and find protocol of Immunoblotting in mybiosource learning center.

How do direct and indirect ELISA differ?

Direct ELISA: Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Indirect ELISA: The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.

Other Frequently Asked Questions

What is a primary antibody?

Primary antibodies are antibodies used in detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease. Primary antibodies bind to target proteins, antibodies or antigens.

What is a secondary antibody?

Antibody that attaches to the primary antibody to detect target antigen is referred as secondary antibody. Secondary antibody have specificity for isotype of primary antibody and often is conjugated. Technically, the secondary antibody’s FAB domain binds to primary antibody’s Fc domain.

Secondary antibodies find application in ELISA, western blot, Immunostaining, Immunohistochemistry and Immunocytochemistry studies.

What is an antibody conjugate?

Conjugated antibody is a monoclonal or polyclonal antibody linked to probe enzyme or agents. It helps in detection in wide range of assay techniques. Also, conjugated antibodies help in detection, purification and microscopy application.

What is the difference between western blot and ELISA?

Both the tests measure immune system’s response to infectious agents. But, ELISA is less sophisticated and cost effective compared with Immunoblotting. The advantage of the Western Blot over the ELISA test is that far more indicators are examined than the single antigen in ELISA.

 What is a western blot?

Western blot is also a detection assay. Unlike ELISA, viral proteins are separated, immobilized and visualized. The proteins are collected into a gel slab and electric current is passed through them. Different proteins based on their size move at different velocities. Once these proteins are segregated by size, the procedure continue like ELISA.

What is ELISA test used for?

ELISA is used quantifying antibody/antigen concentration in viral tests, and as a consumer product ELISA finds application in home pregnancy tests. It is also used in detecting potential food allergens in food products such as milk, peanuts, almonds and eggs. ELISA is used widely in the fields of Immunology, Toxicology and Diagnostics.

Is ELISA test reliable?

As a biochemical assay, ELISA is reliable and effective in detecting antibodies/antigens. In some medical cases, the positive or negative results are always confirmed by Western Blotting. This is mainly done to confirm the test results.

What is the Difference between Direct and Indirect ELISA?

In direct ELISA, the method of antigen mobilization is not specific. When serum is used as the source of target antigen, the proteins in the sample may stick to microtiter plate well and analyte in serum must compete with other proteins to bind with the well surface. This problem is eliminated by using secondary antibody specific for the test antigen in Indirect or sandwich ELISA. This is the key difference between direct and Indirect ELISA.

What is the Difference between Sandwich ELISA and Competitive ELISA?

Competitive ELISA is known for precision and reproducibility whereas Sandwich ELISA is known for sensitivity and specificity. The competitive ELISA is more attractive if no antibody pair can be identified for sandwich ELISA, and when Analyte is too small to bind with a primary and secondary antibody.

What are the variants of Sandwich ELISA?

Sandwich ELISA is known for specificity and sensitivity, but the inherent disadvantage lies in finding antibody pairs to carry out the assay. If the detection antibody is conjugated with an enzyme, the assay is called direct sandwich ELISA and if the secondary antibody is unlabelled, the need for an antibody enzyme conjugate arises, these assays are called Indirect Sandwich ELISA.

What is the difference between Indirect and Sandwich ELISA?

The most popular od ELISAs, Indirect ELISA uses a secondary antibody that binds with primary antibody for detection. The secondary antibody has specificity for primary antibody.

The most powerful in terms of specificity and sensitivity, Sandwich ELISA captures the analyte between two primary antibodies – the capture and detection antibody.

Why is PBS used in ELISA?

Phosphate Buffered Saline is a liquid formulation of buffers and saline. It is used in ELISA procedure to balance PH without disrupting protein binding sites.

What are the commonly used substrates for ELISA?

Horseradish peroxide, Alkaline Phosphatase, β-Galactosidase and Urease. Substrates allow direct visualization and enable kinetic studies.

What is a chromogenic reporter?

Substrates that allow direct visualization and enable kinetic studies are chromogenic reporters. They are far less sensitive compared with fluorescent or chemiluminescent substrates. The detection is assisted with standard absorbance plate readers common to many laboratories. The ease with which one can use chromogenic reporters make them a popular choice as reporting agents.

What are the factors that could affect signal generation in ELISA?

Shape and material of the plate, pH of the buffer, specificity of the capture antibody, incubation time and temperature, conformation and stability of target antigen, specificity, affinity and cross reactivity of detection antibody, concentration and cross reactivity of enzyme conjugate, sensitivity and age of the substrate, and filters and exposure time of the imaging instrument.

Can non-purified antibody be used in ELISA?

Non-purified antibody can be used but may result in higher background. It is highly recommended that purified antibodies be used for optimal signal to noise ratio.

What are some commonly used coating and detection antibodies?

Polyclonal serum, crude ascites, affinity purified monoclonal and affinity purified polyclonal antibody.

What are the recommended secondary antibody concentrations for ELISA?

For Horseradish peroxide, the concentration for colorimetric system should be 20 – 200 ng/ml, the concentration for fluorescent system should be 25 – 50 ng/ml, 10-100 ng/ml for chemiluminescent system.

For Alkaline Phosphatase, 100 – 200 ng/ml for colorimetric system and 40 – 200 ng/ml for chemiluminescent system.

How long does it take to run ELISA?

The assay can be run in less than 3 hours in any formats.

Review on Performance in screening HIV: Rapid Test vs ELISA

Approximately 36 million people are infected with human immunodeficiency virus across the world. In resource-limited settings, there seems to be a migration from ELISA to rapid tests for screening of human immunodeficiency virus. This article is focused on presenting facts that compare ELISA and rapid tests to produce a winner in terms of accuracy.

It is understood there tend to be inconsistencies when two different techniques are compared. Therefore, Western Blot, as most researchers would agree, is the most efficient of all HIV testing techniques, was brought into the experiment to confirm results and compare the performance of ELISA and rapid testing.

In testing HIV, two testing algorithms are used – parallel and serial. In parallel testing strategy, the samples are tested using two different tests i.e. rapid and ELISA. In serial testing strategy, the result of one test determines the need for another. In healthcare centers where serial testing strategy is used, the need for accuracy is paramount so as not to miss infection through false negatives and create confusion through false positives. The screening test is expected to be highly sensitive and the subsequent test is expected to be highly specific to weed out false positives and false negatives.

For this study, 787 samples were tested using both rapid testing kits and ELISA. When samples were analysed by rapid testing kits, 36 samples were found to be reactive, and 40 samples were found to be reactive as per ELISA.

When all the positive samples were processed through confirmatory test, Western Blotting, all the reactive samples obtained through ELISA were found to be reactive as well. Therefore, it is understood, four reactive samples were misdiagnosed as “HIV negative” when tested via rapid testing kits.

When all the reactive samples obtained via rapid tests were processed through the confirmatory test, western blotting, 5 samples were found to be nonreactive. Therefore, in addition to missing four reactive samples and rapid tests misdiagnosed 5 nonreactive samples as reactive.

Implications resulting from false positives and false negatives are huge as it leads to not only confusion but also poor understanding on the part of the patients receiving a reactive report. The false positives of rapid testing kits are often attributed to technical errors, mislabeling of samples, problems with components of the test devices and cross reactivity. In this experiment, both ELISA and Rapid kits were handled by technicians with the expertise to ensure handling accuracy.

Despite high sensitivity, the longer turnaround time of ELISA seems to be the problem for people undergoing HIV testing, whereas rapid tests provide an excellent option for testing immediately. The possibility of missing early infections (false negatives) is a matter of huge concern as the general population is exposed to unwarranted risks.

Following serial testing algorithm across all healthcare centers could be of greater use as preliminary report could be provided immediately (results of Rapid tests) followed by testing of all samples by ELISA to identify any false negative and false positive results. The problems posed by rapid tests can be overcome using ELISA test and western blotting as confirmatory tests. In addition, detection of p24 antigen via rapid tests would significantly enhance efficiency.

References

Rapid Tests versus ELISA for Screening of HIV Infection: Our Experience from a Voluntary Counselling and Testing Facility of a Tertiary Care Centre in North India