Lesson 4c: Detection of viral antibodies

• The detection of newly developed, virus-specific an­tibody or the detection of a rise in titer of preexisting antibody is essential in viral analysis and is without doubt one of the mostly used strategies in epidemiologic research of viruses.
• Most main infections or reinfections consequence within the manufacturing of particular antibodies. As well as, viruses
  corresponding to EBV, HIV, rubella, hepatitis A and B viruses, and arboviruses are troublesome to detect immediately and the serologi­cal analysis stands out as the solely sensible technique of figuring out the actual agent.
• Many different serological techniques have been used in the diagnosis of viral infections.

Detection of Virus antibodies by Hemagglutination Inhibition (HI) assay

• Antibody response to the haemagglutinin protein in the hemagglutinating virus envelope can be measured by the HI test.
• Principle: When serum containing antibodies specific to the hemagglutinating virus are mixed with the virus, the antibodies bind to the haemagglutinin protein in the envelope of the virus blocking the haemagglutinin protein from binding with the receptor sites on the chicken red blood cells.
• Hemagglutination from viruses such as  NDV, Influenza, dengue and ADENO 127 is inhibited when antibodies are present.
• The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum.
• If the serum contains no antibodies that react with the virus, then hemagglutination will be observed in all wells (B)
• Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted (C)
• Purpose: To quantitate serum antibody to a specific avian antigen
• Purpose: To quantitate serum antibody to a specific avian antigen

• By performing two-fold serial dilutions on the serum prior to testing, the concentration of the serum antibodies can be expressed as an HI titre to the log base 2.
• Standardization of the HI test within and between laboratories is very important and is achieved by using (a) a standard 4 HA units virus antigen, (b)standard positive anti-serum and negative serum, (c) a serum control for each test serum to detect the presence of non-specific agglutinins, and by using a standard 1 percent dilution of red blood cells as indicator.
• Materials
  • Thawed serum samples in racks
  • V-bottom microwell plates and covers
  • PBS
  • 1 percent washed red blood cells
  • V-bottom reagent trough
  • 25 µL single and multichannel pipettes and tips
  • Microwell plate recording sheet.
  • Newcastle disease virus antigen diluted to 4 HA units per 25 µL
  • Standard positive and negative serum
• HI Procedure:
  1. Fill in recording sheets to record how samples will be dispensed into microwell plates.
  2. Calculate the number of plates required and number each plate.
  3. Dispense 25 µL of PBS into each well of the plates.
  4. Shake each serum sample and dispense 25 µL into the first well and the last (control) well of a row of a microwell plate.
  5. Use a multichannel pipette to make two-fold serial dilutions along the row until the second last well from the end. The last well is the serum control. Do not dilute this well.
  6. Add 25 µL of the 4HA dilution of antigen to each well excluding the control wells in the last column. See Section 10 for preparation of 4HA units of antigen
  7. Gently tap the sides of the microwell plates to mix the reagents. Cover plates with a lid. Allow to stand for 30 minutes at room temperature.
  8. Add 25 µL of 1 percent washed red blood cells to each well including the control wells in the last column.
  9. Gently tap the sides of the microwell plates to mix the reagents. Cover the plates with a lid. Allow to stand at room temperature for 45 minutes.
  10. Record the pattern observed in each well on a microwell plate recording sheet.
  11. Determine the endpoint. This is the point where there is complete inhibition of hemagglutination.
  12. HI NEGATIVE wells will have a diffuse sheet of agglutinated RBCs covering the bottom
  13. HI POSITIVE wells will have a well circumscribed button of un agglutinated RBCs
• Interpretation of results
  

  In the wells where antibodies are present there will be hemagglutination inhibition. The red blood cells will settle as a button.

   In the wells where antibodies are absent, the red blood cells will agglutinate.

  The end point of the titration is the well that shows complete haemagglutination inhibition.

  Sometimes it is not easy to determine. Look at the size of the button as an indication of the degree of hemagglutination inhibition. Use the control well as a point of comparison. Be consistent in determining the endpoint.

Elution

• The neuraminidase enzyme present in the virus particle will eventually break the bond between the virus and red blood cells. This process is called elution.
• When elution occurs, the red blood cells are no longer agglutinated. They roll down the side of V-bottom microwell plates to resemble the negative settling pattern, a tight button.
• Some Newcastle disease virus strains elute more rapidly and the test must be read before this occurs.
• Usually elution takes longer than 45 minutes. A control well with virus and red blood cells is useful to determine elution time.
• Preparation of 4HA units of virus antigen
• The standard amount of the virus used in the hemagglutination inhibition (HI) test is 4HA units. It is necessary to prepare and test a suspension of the virus containing 4HA units in order to carry out the HI test. This involves a series of following steps.

1. Titrate the stored suspension of virus to be used as the antigen in the HI test.
2. Calculate the dilution factor required to produce 4 HA units. A simple way is to divide the HA titre by 4.
3. Apply the dilution factor and dilute the original suspension of antigen in PBS to produce an adequate volume of 4HA antigen to carry out the HI test. Allow 2.5 mL for each microwell plate.
4. Titrate the diluted (4HA) suspension of virus. This is a back titration to check the diluted antigen contains 4 HA units.
5. Read HA titre. It should equal 4HA units. If not adjust the dilution and titrate again.
6. Use the 4HA unit dilution of antigen in an HI test to test the standard positive and negative serum. The HI titre of the laboratory standard positive serum should equal the predetermined titre.

Detection of Virus antibodies by Neutralization assays

• Serum specimens may be assayed for neutralizing antibody against a given virus by testing serial dilutions of the serum against a standard dose of the virus.
• The anti­body titer is expressed as the highest serum dilution that neutralizes the test dose of virus.
• As a bioassay, neutral­ization assays are highly specific and quite sensitive.
• For many viral agents, the neutralizing antibody level is di­rectly correlated with immunity, an important clinical and epidemiologic endpoint.
• Disadvantages of the assay in­clude the time required to obtain a result and the relatively high cost, due to the labor intensity and requirement for cell culture and titered viral stocks.
• Different neutralization systems include plaque reduction neutral­ization, sometimes using complement enhancement, where the number of virus plaques in control wells are compared with the number seen in cultures inoculated with the virus-serum mixture; microneutralization, an assay performed in microtiter plates requiring small amounts of sera; and colorimetric assays.
• Virus neutralization test (VNT): This test measures the ability of antibodies to block viral infectivity.
• Principle: The virus neutralization or serum Neutralization assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus.  In a neutralization test, serum and virus are reacted together in equal volumes and inoculated into a susceptible animal host or cell culture. If antibodies to the virus are present then clinical disease or CPE will not be observed; that is, the virus replication will be inhibited and virus is neutralized. Thus any assay that measures viral infectivity can be used in virus neutralization.
• Viral neutralization tests are used to either identify an unknown virus using known reference antisera/monoclonal antibody or measure virus neutralizing antibody levels in serum samples against a known infectious virus.
• Specific antibody levels in an animal serum indicate previous exposure to a particular virus and often may give an indication of the susceptibility to a virus.
• Diagnosis of a virus infection requires the submission of paired serum samples: acute phase sample taken at first signs of clinical illness, and convalescent phase sample approximately 2–3 weeks later.
• The virus neutralization test is sensitive and specific, but also more complex, time consuming and expensive than many other assays.
• Procedure

1. Serial dilutions of the serum to be tested are incubated with a standardized quantity of infectious virus
2. After a short incubation period, the serum-virus mixtures are added to cells that support the growth of the virus In parallel with a similar inoculum of virus that has not been incubated with serum is added to the cells. The neutralizing antibody titer is the highest serum dilution that prevents infection of the cells

Interpretation of results

• For positive results, cell will not show any infectivity signs
• For negative results, development of cytopathic effects will be seen in culture

Other methods for Detection of Virus antibodies 

There several other serologic methods which are used for detecting virus antibodies .

These methods are not covered under this topic but I will list them and they might be covered elsewhere. These methods include: Complement fixation tests (CFT), Enzyme immunoassays (Enzyme linked immunosorbent assay (ELISA)), Immunofluorescence techniques (IF), Counter-immunoelectrophoresis, Radioimmunoassays (RIA), Western Blot (WB), RIBA, line immunoassays and Particle agglutination.