Serological methods for virus detection:

 

Serology remains the mainstay for the diagnosis of virus infections in a routine diagnostic laboratory.

The following virus infections are usually diagnosed by serology;

Hepatitis Viruses - hepatitis A, B and C infections are usually diagnosed by serology as these viruses cannot be routinely cultured. Current serological tests including the test for HBsAg are well established and despite the availablity of molecular biological techniques for the detection of viral nucleic acid, serology remains the main means of diagnosis.

HIV - HIV infection is normally diagnosed by serology. The only instance when serology cannot be relied on is in diagnosing HIV infection in the newborn.

Rubella and parvovirus - rubella and parvovirus infections are usually diagnosed by serology. Rubella virus is difficult to isolate and parvovirus cannot be isolated by routine cell culture. The onset of clinical symptomes for these infections coincides with the appearance of antibodies and thus there is little need for other means of diagnosis.

EBV - although EBV serology is reliable, the heterophile antibody test is usually used for diagnosing cases of infectious mononucleosis.
 

The following virus infections may be diagnosed by serology but other means of diagnosis are available. Molecular biology techniques are likely to play an increasing role in the diagnosis of these viral infections.

HSV - although CFT and other serological tests are available for HSV, HSV infections are usually diagnosed by cell culture. Electron microscopy, immunofluorescence and PCR are available as rapid diagnostic methods. Serology is not that reliable in the case of HSV infections, in particular reactivations.

CMV - although serology is available for diagnosing CMV infections, it is not reliable as most cases of CMV infections are a result of reactivation/reinfection. Cell culture (including the DEAFF test) and rapid methods such as the CMV antigenaemia test and PCR are preferred means of diagnosis

VZV - serology can be used to diagnose acute infection and is also used for immunity screening. Cell culture is difficult but a rapid diagnosis may be reached in more severe cases by electron microscopy and immunofluorescence of the vesicle fluid.

Respiratory viruses - diagnosis of respiratory virus infections is more commonly made by cell culture or more rapidly by immunofluorescence of the clinical material. CFT and HAI techniques are usually used for serology and any diagnosis is going to be retrospective.

Enteroviruses - enterovirus infections are usually diagnosed by cell culture. Serology has a very limited role to play as available tests such as neutralization, are cumbersome to perform and in any case, the diagnosis would be retrospective. Serology is of some value in diagnosing cases of coxsackie B myopericarditis as it would be impractical to obtain biopsies of heart tissue.

Rabies - serology is of no value in diagnosing rabies although it may be used to check for immunity after vaccination.

Arboviruses - arbovirus infections may be diagnosed by serology or virus isolation. Arboviruses will not usually grow in routine cell cultures and may require mosquito cell lines or animal inoculation.

The following virus infections are not normally diagnosed by serology, again the role of molecular biology techniques is likely to increase in the near future;-

Diarrhoeal viruses - serology is of virtually no value in the diagnosis of diarrhoeal virus infections as the diagnosis is going to be retrospective. Diarrhoeal virus infections are normally diagnosed by electron microscopy and the detection of viral antigens by ELISA or particle agglutination

Papovavirus - serology is of virtually no value in diagnosing papovavirus infections.

Poxviruses - serology is of little value in diagnosing poxvirus infections.

 

 

 

 

 

 

 

 

 

Serological tests

 

  1. Virus neutralization test
  2. Complement fixation test (CFT)
  3. Hemagglutination inhibition assay (HI test)
  4. ELISA/ELISPOT
  5. Immunoprecipitation and Immunoblotting
  6. Immunofluorescence
  7. Line immune assay

 

1. Virus neutralization:

 

Neutralization of a virus is defined as the loss of infectivity through reaction of the virus with specific antibody. Virus and serum are mixed under appropriate condition and then inoculated into cell culture, eggs or animals. The presence of unneutralized virus may be detected by reactions such as CPE, haemadsorption/haemagglutination, plaque formation, disease in animals. The loss of infectivity is bought about by interference by the bound Ab with any one o the steps leading to the release of the viral genome into the host cells.

 

     Virus neutralization assays are usually conducted by mixing dilutions of serum or monoclonal antibody with virus, incubating them, and assaying for remaining infectivity with cultures cells, embryonated eggs or animals. The end point is defined as the highest dilution of antiserum that inhibits the development of cytopathic effect in cultured cells or virus replication in the eggs or animals.

 

Purpose:        

 

I. Neutralizing antibodies define type-specific antigens on the virus particles.

 

e.g. 3 serotypes of Poliovirus are distinguished on the basis of neutralization tests;

 

Type 1 poliovirus is neutralized by antiserum to type 1 virus but not antiserum to type 2 or 3 poliovirus

 

II. Neutralization tests have been invaluable for virus classification.

III. The knowledge of antigenic structure of a virus is useful in understanding the immune response to viruses and designing novel vaccination strategies.

 

 

Methods:

The antiserum is titrated in the neutralization test against its homologus virus. Serial twofold dilutions of serum is prepared and mixed with an equal volume containing 100TCID50 of virus. The virus and serum mixtures are incubated for 1 hour at 37oC. The time and temperature for incubation varies with different viruses. The mixtures are then inoculated into a susceptible host system. The endpoint titration contains one antibody unit and is the reciprocal of the highest dilution of the antiserum protecting against the virus. Generally 20 antibody units of antiserum is used in the neutralization tests.  

Before neutralization test is performed, one should identify the virus and to identify a virus isolate, a known pretitred antiserum is used. Conversely, to measure the antibody response of an individual to a virus, a known pretitred virus is used. To titrate a known virus, serial tenfold dilutions of the isolate is prepared and inoculated into a susceptible host system such as cell culture or animal. The virus endpoint titer is the reciprocal of the highest dilution of virus that infects 50% of the host system e.g. 50% of cell cultures develop CPE, or 50% of animals develop disease. This endpoint dilution contains one 50% tissue culture infecting dose (TCID50) or one 50% lethal dose (LD50) of virus per unit volume. The concentration of virus generally used in the neutralization test is 100 TCID50 or 100 LD50 per unit volume.

 

2. Complement fixation test (CFT):

 

CFT is convenient and rapid to perform, the demand on equipment and reagents is small, and a large variety of test antigens are readily available. CFT was extensively used in syphilis serology after being introduced by Wasserman in 1909. It took a number of decades before the CFT was adapted for routine use in virology. However, there is now a trend to replace the CFT with more direct, sensitive and rapid techniques, such as RIAs and EIAs.

 

CFT is not as sensitive as a neutralization test or hemagglutination inhibition test. However, it is the first assay performed on sera from infected patients to identify the group to which the infecting virus belongs.

 

 

Purpose:

 

  1. to determine whether antibodies against a virus are present in serum or not.

 

 

Principle:

 

      The interaction of viral antigen and antibody can cause complement fixation, which leads to membrane lysis. RBCs are used as targets because lysis of their membrane is readily observed.

 

If an antigen-antibody reaction takes place, complement fixation takes place. This is detected by adding sheep RBCs that have been coated with rabbit anti-red blood cells antibodies. If complement has been fixed by binding of viral antigen to antibody, the red blood cells will remain intact; if complement is not fixed, the red blood cells will be lysed by the action of free complement.

    

Method:

a. Titration of hemolytic serum and complement

Dilutions of complement with 20% difference in concentration are made from 1:30 to 1:279. The following dilutions of hemolytic serum are made: 1:400, 1:800, 1:1600, 1:2000, 1:2400, 1:2800, 1:3200.

The following controls are required:

  1. cell control - unsensitized cells only
  2. complement control - complement at different concentrations and unsensitized cells
  3. haemolytic serum control - sensitized cells only at different concentrations of haemolytic serum

b. Titration of antigen and antibody

Antigen at dilutions of 1:2 to 1:512 is titrated against positive serum control. The following controls are incorporated:

  1. antigen control - antigen at different concentrations, complement and sensitized cells
  2. antibody control - antiserum at different concentrations, complement and sensitized cells
  3. cell control well - sensitized cells only
  4. complement back titration

The optimal dose of the antigen is the highest dilution of antigen that gives 75% or more fixation with the highest dilution of antibody.

c. CFT proper

In the CF proper, the haemolytic serum is used at the optimal sensitizing concentration, the complement at 3HD50, and each individual antigen at the optimal dose. Patients' sera should be inactivated at 56oC for 30 minutes before.

(Why patients’ serum has to be inactivated at 56oC for 30 min? To inactivate complement present in patients’ serum).

The following controls should be present;

  1. Serum control - serum and complement only, to detect any anticomplementary activity in the serum
  2. Antigen control - antigen and complement only, to detect any non-specific reaction between antigen and complement.
  3. Complement back titration - to check that the complement is used at the correct strength
  4. Cell control - sensitized cells only, to check that the cells were suitable for use.

All controls should show complete lysis and the highest dilution of patient serum that still shows a reading of 3 or 4 is the CF titer. Diagnosis of a recent infection is usually made by the detection of a fourfold or greater increase in titer or by the detection of a high antibody titer from a single specimen (1:80 or above).

Complement Fixation Test in Microtiter Plate.

Advantages of CFT

  1. Ability to screen against a large number of viral and bacterial infections at the same time.
  2. Cheap

Disadvantages of CFT

  1. Not sensitive - cannot be used for immunity screening
  2. Time consuming and labor intensive
  3. Often non-specific e.g. cross-reactivity between HSV and VZV.

 

 

 

 

 

 

 

IV. Hemagglutination Inhibition test (HI):

It is called hemagglutination inhibition because it measures the ability of soluble antigen to inhibit the agglutination of antigen-coated red blood cells by antibodies.

Purpose:

-         to quantitate soluble antigens and HI titer in serum samples

-         sensitive than CFT, simple, inexpensive, and rapid and is the method of choice for assaying antibodies to any virus that causes hemagglutination

-         commonly used for different  strains of influenza viruses, and parainfluenza viruses

-         Hemagglutinins are used as antigens in influenza virus vaccines, thus making HI the method of choice for measuring vaccine-induced antibodies

Principle:

If antibodies against the hemagglutinins are present, hemagglutination will be prevented. In HI test, serial dilutions of serum are mixed with a known amount of virus. After incubation, RBCs are added, and the mixture is left to sit for several hours. If hemagglutination is inhibited, a pellet of RBCs forms at the bottom of the tube. If hemagglutination is not inhibited, a thin film is formed. The HI titer of the serum is the reciprocal of the highest dilution at which hemagglutination is prevented.

In this test, a fixed amount of antibodies to the antigen is mixed with a fixed amount of red blood cells. By serially diluting the sample, one can quantitate the HI titer of unknown sample.

Methods:

1.      0.05 ml PBS is dispensed into appropriate micro titer plate wells

2.      Perform serum dilution in the range of 1:10 to 1:1280 in PBS

3.      0.05 ml of antigen (4HA units/0.05ml) is added to each well

4.      Plate is incubated at room temperature for 1 hour

5.      0.025 ml of 0.5% bovine RBC is added to appropriate wells

6.      Plate is incubated at 4oC overnight (18-20 hrs).

7.      The highest dilution of serum completely inhibiting HA is considered the HI titer of that serum.

 

 

 

 

V. Line immunoassay (and/or Recombinant immunoblot assay)

Line immunoassay is used for the detection of antibodies to virus in human serum or plasma. It is intended for use as a supplementary test on human serum or plasma specimens found to be reactive in an anti-HCV, anti-HIV or anti-HTLV screening procedure.

Purpose:

-         Highly sensitive, specific and confirmatory test

-         The test is simple and easy to perform. Its interpretation can be made visually by means of a user friendly reading chart.

-         Can be used for many viruses e.g. hepatitis C virus, HIV, HTLV and T. pallidum also.

Test principles:

A nylon strip has multiple different individual antigens attached in bands at different points along the strip. When serum is applied and if there are specific antibodies, they will bind to their respective antigens at different points on the strip. After a second step in the process, a colored band is developed for each individual antibody in the serum.

The idea behind this test is that it is possible that one might get a positive reaction in an ELISA or a positive reaction to one antigen on the strip purely by a chance cross-reaction. If there are antibodies to several different individual antigens of the micro-organism; this is not likely to have occurred by chance.

Principle [(with an example of HCV detection) INNO-LIA HCV Score assay)]

Strip design




INNO-LIA™ HCV Score

The INNO-LIA™ HCV Score assay utilizes well-defined antigens derived from HCV immunodominant proteins from the core region, the E2 hypervariable region (HVR), the NS3 helicase region and the NS4A, NS4B and NS5A regions. The antigens used are either recombinant proteins or synthetic peptides, highly purified, and fixed on a nylon membrane.

In addition the strip includes four control lines: a streptavidin control line, weak and medium positive control lines (human IgG), and a strong positive control lines (anti-human IgG) which is also the sample addition control line.

 

A rating, from "-" to "4+", is given to the antigen lines on the developed strips by comparing their intensities to those of the control lines.

 

Intensity of antigen lines (R) compared to control lines

Rating

Lower than +/-

R <+/- -

Equal to +/-

R =+/- +/-

Higher than +/- and lower or equal to 1+

+/- < R< I+ 1+

Higher than 1+ but lower than 3+

1+ < R < 3+ 2+

Equal to 3+

R = 3+ 3+

Higher than 3+

R > 3+ 4+

 

 

 

 

 

 

 

 

 

 

Terminologies in virology:

I. TCID50 (Tissue culture Infectious dose 50)

The quantity of virus in a specified suspension volume (e.g., 0.1 ml) that will infect 50% of a number (n) of cell culture microplate wells, or tubes, is termed the Tissue Culture Infectious Dose 50 (TCID50).

If the TCID50 is equivalent to a virus titer of 10m infectious doses, then log TCID50 = m. This expression for the '50% endpoint dilution' is also applicable to antibody titers measured in neutralization tests.

In a test to measure the 50% endpoint dilution of, for example, a virus suspension, individual cell monolayers are inoculated with a dilution series of the original suspension. Each dilution is inoculated onto n separate cultures, and observations are made of the number of ensuing infected monolayers (as a proportion of n) for each dilution.


II. EID50 (Egg infective dose 50)

The EID50 or Egg Infective Dose 50% is the dose required to establish infection in 50% of the inoculated eggs. Influenza viruses can be cultured and assayed by inoculating the virus into fertilised chicken eggs.

In the study of Avian Influenza, the quantification of virus shed from infected birds is valuable in pathogenesis studies and to determine the effectiveness of vaccines, and is performed routinely by cultivation of virus containing samples using embryonating chicken eggs (ECE) and expressed by 50% egg infectious dose (EID50). Although, this assay is accurate and is the standard test for infectious virus titration, the method is laborious, requires a large number of ECE, and takes at least 7 days to determine results.

 

III. MOI (Multiplicity of infection)

MOI is defined as ratio of infectious virus particles to cells.

It is important that the cells not overgrow the culture or there won't be sufficient medium resources to dedicate to virus or protein production. On the other hand, it is important that there not be so much virus present that the cells are killed before they can expand into the culture and utilize all its resources. Ideally, the virus will stop the cell number between about one third and two thirds the maximum density obtained with your cell line in the absence of the virus.

 

Why use low MOI?

Low MOI is the best strategy for expanding or amplifying virus stocks. This is for two reasons. Expanding at low MOI provides the quickest and easiest way to rapidly expand the volume of virus that you have. In addition, low MOI amplification is the best strategy for preserving the genetic integrity of your virus and preventing the buildup of DIPs (Defective Interfereing Particles that represent partial genomes packaged by complementation from intact genomes coinfected in the same cell).

Low MOI infections can also be used to produce protein, but they are more difficult to control and less reliable for producing maximal amounts of protein. They do have the advantage, however, of requiring much less virus stock. This can be a great advantage for large scale production, provided they can be reliably reproduced.

 

Why use high MOI?

In general, High MOI infections are to synchronously infect all the cells in the culture with at least one virus particle. The probabilities of this process are generally described by the Poisson distribution, but the short answer is that if you want all the cells infected simultaneously; infect the culture at an MOI near 3. Since the cells are not going to grow anymore once you've added the virus you can precisely control the cell density at infection to hold around one half of maximum stationary phase density.

 

IV. Plaque forming units (PFU/ ml)

(Look at Virus isolation and Virus titer handouts).