22 - Detection and titration of antiviral antibodies

Question 1

Which methods can virus demonstration be divided into?

Give the advatages and disadvatages

Answer 1

1. Virus demonstration
   
   • Direct method
   • ​Advatage: isolation of the causative agent within short time (in case of NA/Ag detection)
   • Disadvantage:
     
     • Can only be performed for few days
     • Expensive
2. Serological methods
   
   • Indirect method
   • ​Advantage:
     
     • igher chance of demonstration (longer persistance of antibodies)
     • Cheaper
   • Disadvantages: can not differentiate between maternal/vaccine/seroconversion antibodies
     
     • Hence must consider maternal immunization/antibodies
   • Aim:
     
     1. Survey - viruses in stock → random sampling
     2. Individual diagnosis → identification of the causative agent → paired sera (serum titer) (se figur)

Question 2

List the different types of serological tests

Answer 2

• Virus neutralization (VN)
  
  • ​All cytopathogen viruses ⟷ non cytopathogen viruses
• Hemagglutination Inhibition Test (HAI)
  
  • Haemagglutinating viruses (PI-3, NDV, influenza, etc)
• ELISA
  
  • ​Marker vaccines (IBR, pseudorabies)
  • Competitive (multispecies kits
  • Indirect sandwich
• AGID
  
  • ​Adenovirus, Maedi-Visna, bluetongue
• CF
  
  • ​FMD, EIA

Question 3

Virus neutralization

Principle

Answer 3

• Main antibody test
• Heat inactivates serum to eliminate non-specific ABs. Blocking antibodies around virus means virus can not attach to cell receptors → no CPE
• In the presence of blocking antibodies reacting with anti- receptors of viruses the virus is not able to adsorb to the cells
• Serotype (species) specific

Question 4

Virus neutralization

Name the methods of virus neutralization

Answer 4

1. Constant virus varying serum dilution method
2. Constant serum varying virus dilution method

Question 5

Virus neutralization

Constant virus varying serum dilution method

Answer 5

• For antibody detection
• Method:
  
  • Serial twofold dilution form the sera
  • Incubation 1hour in 37ºC → antibodies neutralize the virus
  • Inoculation of the cell -cultures with each dilution
  • Incubation 37ºC for several days
• Results:
  
  • Any CPE proves no blocking antibodies are present

1. Serum neutralising titer
   
   • Greatest dilution of serum that neutralizes the virus (to 50% CPE)
   • Hence animal protected! – Only test to prove this – is an animal protected?
   • Test validity – A virus between 30-300 TCID
2. Plaque reduction test
   
   • ​Fewer plaques compared to control viruses
   • Proves blocking antibodies
   • Serum Neutralization Titer:
     
     • The greatest dilution of serum so that fewer plaques occur than in the control
       
       • E.g. if Virus Control has 10 plaques – If Serum = 4 plaques then reduction 60%
     • Termed “Sera positive” e.g. Rabies VN – 50%; Equine Arteritis VN – 75%

Question 6

Virus neutralization

Constant serum varying serum dilution method

Answer 6

• Neutralisation index calculation → for virus identification

Steps:

• 2 serial tenfold dilutions of the given virus suspension
• Adding negative and positive serum
• Incubation 1^h 37ºC
• Inoculation of the cell-cultures from each dilution
• Incubation 37ºC for several days
• CPE

Evaluation:

• If quotient equal or greater than 2, then the virus is the same as in the positive serum
• Evaluation – If quotient equal or greater than 2 then virus is same as in positive serum

Question 7

Hemagglutination inhibition

Answer 7

• Antibody test
• Only for hemagglutinating viruses
• Exhausting of the sera 37ºC, 1 hour
  
  • Eliminates the non-specific hemagglutinating activity

Method:

1. Serial twofold dilution of the serum sample
2. 4-8 hemagglutinating units added to each serum dilutions
3. Incubated 1 hour, at virus specific temperature
4. Adding washed erythrocytes (1%)

Result:

Hemagglutination shows virus not inhibited by antibodies hence no antibodies

Serum hemagglutination inhibition titer:

• The highest dilution where there is no hemagglutination (enough antibodies to block the hemagglutination proteins)
• Validity of the test:
  
  • A virus between 4-8 HAU
  • Actual titer of the known positive serum did not change more than one dilution level since the previous test