• The presence of viral antigens in clinical specimens, such as nasopharyngeal aspirates, fecal specimens, vesicle fluids, tissue specimens, as well as serum samples can be demonstrated by antigen detection assays.
  • Methods for the detection of virus-specific antigens have allowed rapid identification of a wide variety of viruses.

Detection of Virus antigens by Hemagglutination assay (HA)

The ability to agglutinate erythro­cytes, a property shared by many viruses, can be used for the identification of some of these viruses. Hemagglutination is visible macroscopically and is the basis of hemagglutination tests to detect the presence of viral particles. The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells.

Principle: Some viruses such as Newcastle disease virus and Influenza viruses have hemagglutinin (HA) proteins on their surface which tends to bind to sialic acids on the surface of red blood cells (RBCs) leading to agglutination. The attachment/binding of viral particles by their receptor sites happens to more than one cell. As more and more cells become attached in this manner agglutination becomes visible.

  • Normally, red blood cells will fall to the bottom of a culture well, forming a sharp dot (A)
  • However, if viruses are present, the RBCs become bound to the virus particles in a lattice or network (B)
  • Only U and V shaped wells can be used, not flat bottomed flasks
  • HA can be used as the basis for quantifying the amount of viruses that possess hemagglutination property (eg, influenza A virus). HA cannot accurately determine the number of virus particles present in a sample (ie, virus particles/mL); but it is useful for comparing the relative concentrations of a virus between samples, such as those obtained from multiple infected hosts, or those collected sequentially from an individual host on different days or times.
  • Examples of hemagglutinating viruses: Influenza viruses (i.e Avian influenza virus), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Adenovirus II.

Note: virus is NOT infecting RBCs, just binding and there are some other viruses and some bacteria which will also agglutinate chicken red blood cells

  • HA Procedure
  • HA is done by carrying out two-fold serial dilutions of the viral suspension in a microwell plate and then testing to determine an end point.


  •                                                                                                                      Titer = 32 HA units/ml
  • Briefly, dispense 25 µL of PBS into each well of the microwell plate. Place 25 µL of test samples in first well of each row of column 1. Samples can be tested in duplicate or triplicate if necessary. Use a multichannel pipette to carry out two-fold serial dilutions across the plate until Column 11. See Appendix 4 for instructions on carrying out two-fold serial dilutions. Add 25 µL of PBS to each well. Add 25 µL of 1 percent red blood cells to each well including Column 12. The wells in this column are control wells that contain only PBS and red blood cells. Gently tap sides of the plate to mix. Place a cover on the plate. Allow the plate to stand for 45 minutes at room temperature. Read and record the results in each well. All the control wells should be HA negative.
  • HA negative: A sharp button of red blood cells at the bottom of the V-bottom well.
  • HA positive: A hazy film of red blood cells, no button or a very a small button of red blood cells at the bottom of the V-bottom well.
  • Identify the end point. This will be the last well to show complete hemagglutination and contains one hemagglutinating unit.                                                                                                                

Reading and Interpretation of results

The HA plate performed for Newcastle disease virus where by on the left are samples labelled 1 to 7 with the negative control at the last row while on the right side there are HA titers for each sample.

  • 1 HA unit: One HA unit in the hemagglutinin titration is the minimum amount of virus that will cause complete agglutination of the red blood ells. The last well that shows complete agglutination is the well that contains one HA unit.
  • The end point is the well with the lowest concentration of the virus where there is hemagglutination (that is where the titer is read)
  • The HA titre or Titer is the maximum dilution that gives visible agglutination. In other words, “HA titer” of the stock virus sample is reported as the inverse of the highest dilution that completely agglutinates the red blood cells.
  • In the figure above, sample 2 has a titre of 64 = 26 which is the dilution of 1 in 64 (1/64). This dilution (1:64) contains 1 HA unit (one haemagglutinating unit).

 The HA titre of the test sample is therefore the reciprocal of 1/64 = 64 = 26

 The titre of the suspension of Newcastle disease virus can be expressed as 64 or 26 HA units in 25 mL.

Preparation of RBCs used for HA assay is found here


Rapid hemagglutination test

This test can determine the presence of a hemagglutinating agent in one minute. If testing many samples at the same time, it is necessary to test the negative and positive control samples only once.


  • Clean glass microscope slide or a clean white ceramic tile.
  • 10 percent suspension of washed chicken red blood cells. See Section 8.
  • Micropipette and tips, glass Pasteur pipette or a wire loop.
  • PBS.
  • Negative and positive control allantoic fluid samples.
  • Sample to be tested for the presence of Newcastle disease virus, for example allantoic fluid.


1. Place 4 separate drops of 10 percent chicken red blood cells onto a glass slide or a white tile.

2. To each drop of blood, add one drop of the control and test samples as follows. Use separate tips, pipettes or a flamed loop to dispense each sample.

Drop 1 PBS

Drop 2 Negative control allantoic fluid (no haemagglutinin)

Drop 3 Positive control allantoic fluid (contains haemagglutinin)

Drop 4 Unknown sample to be tested

3. Mix by rotating the slide or tile for one minute.

4. Observe and record results. Compare results of the test samples with the control samples.


 Agglutinated red blood cells in suspension have a clumped appearance distinct from non-agglutinated red blood cells.

 The red blood cells mixed with the positive control allantoic fluid will clump within one minute.

 The red blood cells mixed with the PBS and negative control allantoic fluid remain as an even suspension and do not clump.

 Judge the results of the test sample by comparison with the positive and negative controls.

 The PBS and negative allantoic fluid controls are used to detect clumping of the red blood cells in the absence of virus. This is unlikely to occur. If it does occur, the test is invalid.

Detection of Virus antigens by Immunofluorescence (IF) techniques

  • Principle: In immunofluorescence tests, cells from a clinical specimen are fixed on a glass slide and viral antigens present in the cells are detected by fluorescein-labelled virus-specific antibodies.  Once antibody is labelled with a fluorochrome, the antigen–antibody complex emits light of a particular longer wavelength when excited by short wavelength light. This can be visualized as fluorescence in an ordinary microscope after light of all other wavelengths is filtered out.
  • Solubilized antigens in clinical specimens are first captured using specific monoclonal antibodies bound to a solid phase, and are then detected with virus-specific detector antibodies.
  • Immunofluorescence (IF) or fluorescent antibody staining is an antigen-detection test that is used primarily on frozen tissue sections, cell “smears,” or cultured cells; formalin-fixed tissue samples are generally not useful with this procedure.
  • Antigen is detected through the binding to the sample matrix of specially modified, agent-specific antibodies.
  • The modification is the “tagging” of the antibody with a fluorochrome that absorbs ultraviolet light of a defined wavelength, but
    emits light at a higher wavelength.
  • The emitted light is detected optically with a special microscope equipped with filters specific for the emission wavelength of the fluorochrome.
  • The fluorochrome can be bound directly to the agent-specific antibody (direct immunofluorescence) or it can be attached to an anti-immunoglobulin molecule that recognizes the agent-specific antibody (indirect immunofluorescence).
  • Immunofluorescence tests are also termed as fluorescent antibody test (FAT)

           Direct Immunofluorescence

  • For direct immunofluorescence, a frozen tissue section, or an acetone-fixed cell smear, or monolayer on a coverslip, is incubated with fluorescein-tagged antiviral antibody.
  • Unbound antibody is then washed away, and the cells are viewed by light microscopy using a powerful ultraviolet/blue light source.
  • The apple-green light emitted from the specimen is revealed (against a black background) by incorporating filters into the eyepieces so that all the blue and ultraviolet incident light is adsorbed.

The direct FA test for rabies-suspect brain tissue. The fluorescently-labelled anti-rabies antibody incubated with rabies-suspect brain  tissue, binds to rabies antigen. Unbound antibody can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope. If rabies virus is absent there will be no staining.

          Indirect Immunofluorescence

  • Indirect (“sandwich”) immunofluorescence differs in that the antiviral antibody is untagged, but fills the role of the meat in the sandwich.
  • It binds to antigen and is itself recognized by fluorescein-conjugated anti-immunoglobulin.
  • The high affinity of avidin for biotin can also be exploited in immunofluorescence, by coupling biotin to antibody and fluorescein to avidin.
  • In the diagnostic setting, immunofluorescence has proved to be of great value in the early identification of viral antigens in infected cells taken from patients with diseases known to have a relatively small number of possible etiological agents.
  • There is little difficulty in removing partly detached infected cells from the mucous membrane of the upper respiratory tract, genital tract, eye, or from the skin, simply by swabbing


Detection of virus antigens by Immunocytochemical techniques

  • Immu­nocytochemical staining is a sensitive and specific method for detecting viral antigens with labelled antibodies.
  • This technique was pioneered by Coons in 1942 for studying the structure and function of a variety of viral proteins and continues to be utilized in both the research and clinical laboratories.
  • It has been used both for detec­tion of viral infection of a monolayer prior to the appear­ance of cytopathic effect and in rapid screening assays for drug resistance.
  • This method utilizes reagents simi­lar to those used in the IF assay except that the fluorescent marker is replaced by an enzyme.
  • When enzyme-specific substrates are provided, a coloured precipitate forms at the site of reaction.
  • Typical enzymes used to detect viral antigens include alkaline phosphatase and horseradish peroxidase.
  • A major drawback of alkaline phosphatase­ based reagents is their lack of stability; a major drawback
    of peroxidase as a marker is the fact that this enzyme is endogenous to some mammalian tissue, thus requiring
    either elimination of the endogenous enzyme or use of a nonmammalian enzyme, glucose oxidase.
  • Advantages of immunoenzymatic staining compared to IF staining include the virtual permanence of stained preparations and the ability to view slides using an ordinary light microscope.

Detection of Virus antigens by Radioimmunoassay(RIA) techniques

  • The first important use of RIA in diagnostic virology was for the detection of hepatitis B surface antigen.
  • The origi­nal RIA described by Yalow and Berson was a com­petitive binding assay in which the competition between an unlabelled antigen and a radiolabelled antigen reacting with a limited amount of antibody over a short period of time was monitored.
  • There are two types of RIA methods: the direct and indirect solid-phase RIA.
  • In the direct solid-phase RIA, antigen or antibody are captured on a solid support and detected by radiolabelled (usually I25J) antibody or anti­gen, respectively. The amount of signal increases propor­tionally to the amount of antigen or antibody present in the sample.
  • In the indirect assays, the capture of antigen or anti­body to the solid phase prevents the binding of labelled antibody or antigen, respectively, so that the amount of signal detected is inversely proportional to the amount of antigen or antibody present.
  • For the most part, RIA methods have been replaced by EIA for routine diagnostic purposes due to the complexity of the assay, the use of radioisotopes, lack of standardized commercially avail­able reagents, and high equipment costs.

Detection of Virus antigens by Enzyme immunoassays (EIAs) techniques

  • The assays used in this method rely on anti­bodies directed against a specific virus or viral antigen that are adsorbed or directly linked to polystyrene wells in microtiter plates, plastic beads, or membrane-bound material.
  • When viral antigen is present in a specimen, it binds to the immobilized antibody and a second “detecting” antibody conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase then attaches to the antigen, forming a three-layer “sandwich” consisting of the immobilized antibody, the antigen, and the detecting antibody with enzyme attached.
  • A substrate specific for the enzyme is added and a colour reaction occurs that can be monitored by spectrophotometry or by direct visualiza­tion.
  • The test is quite simple to run, requiring only stan­dardization of reagents and techniques such as dilution, incubation, and washing.
  • EIA test has the distinct advantages of being simple to perform, utilizing reagents that have long shelf
    lives, are inexpensive, and do not require sophisticated technical evaluation to determine results.
  • EIA technique is also sensitive (less than 1 ng/ml), specific, rapid, safe, with automation potential, and low
    cost, particularly when many specimens require evalu­ation.