ELISA
and Western blot testing
Appreciating Giraldo’s conclusions requires
an understanding of the ELISA and Western blot techniques.
Both ELISAs and Western blots detect proteins,
either antibodies or antigens. Antigens are foreign proteins, such
as those belonging to viruses, which the immune system targets for
destruction. One way that immune systems destroy antigens is by
producing antibodies that lock onto, or "neutralize," them. A particular
virus may contain about ten different proteins that get exposed
to the immune system. Each one of these proteins triggers the production
of different species of antibodies, just as a door with ten bolt
locks requires ten different keys.
ELISA stands for Enzyme-Linked Immunosorbent
Assay. Special enzymes linked to the test proteins luminate at intensities
according to the amount of targeted protein that exists in the serum.
Western blots work similarly. But, unlike ELISAs, which hold the
different species of test proteins together, Western blots separate
the different test protein species into different bands, according
to their molecular weights. Thus, whereas a positive ELISA indicates
that serum contains target proteins that react with at least one
test protein, it cannot determine how many or which species of test
proteins have reacted. Western blots can.
(The word "Western" honors the scientist
who developed the technique, first used for DNA. The DNA procedure
is called the "Southern blot," after the scientist’s
last name, and when employed for RNA, the procedure is called the
"Northern blot.")
These tests are inexpensive, easily performed
substitutes for the only absolute method of determining if a person
is actively infected with a microbe: isolation of the microbe from
fresh patient tissue. In the case of a microbe that infects immune
cells, which is what HIV is said to be, that would mean isolating
HIV from fresh blood.
The accuracies of these tests are determined
by successfully obtaining positive results in people from which
isolations can be obtained (sensitivity), and successfully obtaining
negative results in people from which isolations cannot be obtained
(specificity).
Establishing test
parameters
Though reactivity of a single target protein
earns a positive ELISA designation, a positive Western blot may
not require that every target protein react–not if microbial
isolation data shows that a certain combination of positive reactions
corresponds to a maximum accuracy in identifying people who are
and who are not actively infected with that microbe.
Giraldo emphasizes a point that most HIV
professionals overlook, and which plays a salient role in his investigation:
ELISAs and Western blots are not only qualitative (they indicate
if the target proteins are present in the serum), they are also
quantitative (they indicate how much of the target proteins are
present in the serum). Each can measure the amount of target proteins
in the sera by the intensities of the test reactions, as determined
by their observed luminosities.
ELISA and Western blot test instructions
stipulate what luminosity level constitutes a positive reaction,
and that the level varies according to the microbe being tested
for. That opens the question: At what level of luminosity should
a reaction be regarded as "positive"? The answer, as always, lies
with isolation of the microbe. Only isolation data can logically
determine the ELISA and Western blot luminosity levels that most
accurately distinguish who has or doesn't have an active infection
with a particular microbe.
ELISAs and Western blots can test for either
antigens or antibodies, depending on what the test kit contains.
Antigen tests contain antibodies and react if the serum contains
antigens (the actual virus proteins, in the case of a viral test);
antibody tests contain antigens and react if the serum contains
antibodies. The HIV antibody tests, then, contain presumed HIV proteins,
which are the viral antigens. They react with sera that possess
antibodies that neutralized these antigens. What is called "the
HIV test" consists of a battery of sequentially administered antibody
tests, two ELISAs followed by at least one Western blot.
Both antibody and antigen tests can be reliable
and valid indicators of viral infections. But only virus isolation
can demonstrate if either accurately identifies who has and who
doesn't have an active viral infection.
ELISA and Western blot tests exist for HIV
antibodies and for HIV antigens. But the HIV antigen tests are not
used for diagnosing HIV infections. Like the questions Giraldo has
asked about the unusually high dilution levels for HIV tests, nobody
has ever explained why HIV-antigen tests are not used to diagnose
HIV infections. But the technical literature is very clear: while
many members of the risk groups, including most who have "AIDS"
conditions, test HIV-antibody positive, only those with "AIDS"
conditions tend to test HIV-antigen positive as well (Piatak, Science
259, 1993). So whereas HIV antibody tests identify as positive lots
of healthy people, antigen tests do not.
Giraldo says that even diluting of serum
can be a valid practice and produce reliable results. But, again,
only if the diluting has been established by isolation to improve
accuracy.
– P. P.
--Paul
Philpott