Page 189 - TBE_Book_V2_2019

Chapter 10: Diagnosis

Indirect detection of TBEV flaviviruses and can also give positive results
for some time after a TBE vaccination. The CFA
infection relies on the quality of the reagents used
being excellent, especially the TBEV antigen
Purified antigenic components of the TBEV (which was formerly mouse brain extract but
particle are essential in order to be able to
extracts from infected cell cultures were
detect antibodies produced by a potential subsequently used). The introduction of
host. The main immunodominant structure of modern, standardized, less time-consuming
a TBEV particle is the dimeric envelope (E) assays and the lack of antigen of appropriate
protein, which induces hemagglutinating, quality means that the CFA is now obsolete.
neutralizing, and protective antibodies
following infection or immunization. The Hemagglutination inhibition test
capsid (C) protein and nonstructural protein 1
(NS1) are antigens against which the host The hemagglutination inhibition (HI) test
generates complement-fixing antibodies. A exploits the ability of the E protein of TBEV
more detailed description of the proteins and other flaviviruses to agglutinate
encoded by the TBEV genome can be found in erythrocytes isolated from male geese. The
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Chapter 2b. agglutinating phenotype of the TBEV is lost in
the presence of host antibodies against the E
Complement fixation assay protein and only a small pellet of erythrocytes
forms at the bottom of the test tube, whereas
The complement fixation assay (CFA) is one of a larger layer of erythrocytes can be seen to
the oldest tests for detecting antibodies form at the bottom of the tube in the absence
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against TBEV and other flaviviruses, and was of host anti-virus antibodies. The test can be
used to detect anti-virus antibodies in the standardized using a defined quantity/activity
early phase of a potential infection. The CFA of antigen (usually 4 hemagglutination units),
cannot differentiate between different a defined concentration of erythrocytes, and
antibody isotypes, however, because IgM and serial dilutions of the serum being tested. The
IgG (IgG1, IgG2, and IgG3 subclasses) can all test can therefore be quantitated and the
bind complement. Early data showed that
level of dilution at which the serum inhibits
infected individuals display a marked increase agglutination is referred to as the HI titer. It
in the generation of complement-fixing
should be noted that serum contains many
antibodies during the second phase of the substances that inhibit hemagglutination and
infection involving CNS symptoms, about 10- these must be removed by acetone extraction
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14 days after being infected. The titer of
or kaolin absorption before the serum can be
complement-fixing antibodies reaches a peak used in the HI test. Usually the viral antigen
after 5-10 weeks and then decreases to a
used in the test is isolated from infected
lower level or disappears completely following mouse brain, although cell culture super-
a period of up to 1 year. The detection of natant can also be used as a source of antigen
complement-fixing antibodies is therefore an
when testing for other viruses.
indicator of an acute or recent TBEV infection.
The test usually involves demonstrating a The hemagglutination reaction detects both
significant increase in antibody titer in 2 serum IgM and IgG antibody isotypes. Historically,
samples taken 10-14 days apart. During the the HI test was used to demonstrate a
acute phase of the disease, a 3- to 4-fold significant (usually 4-fold) increase in the end
increase in titer may be expected. The CFA is titer that would be indicative of an acute
cross-reactive with antibodies against other infection.

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