ANTIBODY TESTS AND ELISA IN HIV; IS IT
TOO OLD?
HIV
or human immuno-deficiency virus was initiated as a silent epidemic in 1981.
Since its inauguration, the virus has claimed millions of lives worldwide. HIV
has been classified as HIV-1 and 2. HIV-1 is more pathogenic with a higher
prevalence rate than HIV-2. Most HIV patients lack symptoms but may be
subjected to a flue like illness with a rash and therefore early detection
methods must be made extra sensitive to control and prevent spread of the disease
which will help patients respond to therapeutics. HIV antibody tests such as western blot,
nucleic acid based tests, polymerase chain reaction [PCR] and IFA
[immuno-fluorescence assay] may be used in the identification of the disease.
However, the most commonly used method is ELISA or enzyme-linked immune-sorbent
assay (WHO, 2002).
ELISA
is commonly used to detect both primary and secondary antibodies [PA and SA] to
HIV-1 using samples of blood, saliva or urine and is an appropriate diagnostic
test for adults. ELISA is created through protein antigens which are synthetic
peptides that identify and characterise HIV epitopes, using qualitative and
quantitative detection techniques in the measurement of either the antigen or
antibody. The principle of ELISA is based on the use of enzymes such as
alkaline phosphatase or β-galactosidase which is conjugated with a PA and SA
usually 1:1000 to 1:2000 dilutions respectively by phosphate buffered saline
[PBS], a colourless substrate generating a coloured chromogenic product
(Winkler, 2005). Homogenize tissue is usually centrifuged at 10,000 rpms for 20
minutes with 0.1ml of supernatant in each well for 12 hours followed by washing
of the microtiter plate comprising of 96 wells using PBS for 3 minutes. The
next step is the addition of 0.1ml of PAs for 1-2 hours at room temperature
followed by a PBS washing and an addition of 0.1ml of SAs and a further washing
using PBS. The final step is the addition of a dye such as alkaline phosphatase
yellow [pNPP] with 0.05ml of sodium hydroxide followed by the measurement of
optical density [OD] using absorbance values obtained from a spectrophotometer.
The OD values produced are based on the absorbance of light by the sample which
is proportional to the antibody concentration. Furthermore, a mathematical
calculation is used to obtain a cut off point to OD using negative controls
that are multiplied by a factor. Samples consisting of OD values of ≥ 0.1 are
said to be positive for HIV infection (Hongbao et al 2006). ELISA provides a
positive result in the presence of HIV antibodies and is available within 2-4
days. However, if the test is conducted during the seroconversion period the
test will provide negative results. Uncertain ELISA results may occur during
the presence of autoimmune diseases such as leukaemia or syphilis and the use
of corticosteroids indicating interference from other antibodies and may
require PCR tests. Abnormal ELISA results may occur due to pregnancy,
haemodialysis and contamination in the laboratory while false positive results
may occur amongst hepatitis B patients (Wai et al 2002). Although, the FDA has
approved only 10 ELISA test kits there are about 40 unlicensed kits available.
ELISA is classified as indirect, competitive, sandwich and modified and uses various
molecules. However, the most common type of ELISA in the identification of HIV
antibodies is sandwich (Hangboa et al 2006).
Sandwich
ELSIA on the other hand is the measure of antigens where antibodies are
immobilized on a microtiter well. Upon the addition of the sample containing
antigens the antibodies bind to the antigens. The antibodies in the sample
become sandwiched between 2 antigen molecules with 1 immobilized within the
solid phase and the other containing the enzyme. This is followed by washing
and the addition of a secondary enzyme linked antibody specific for a different
epitope on the antigen and is allowed to react with the bound antigen. The next
step involves the washing of SAs and the addition of the substrate resulting in
a coloured product proportional to antibody concentration which is then
measured. This method may be the most sensitive in the identification of all
iso-types of antibodies of HIV although, a relatively large sample volume [150
µL] is required hence making repeat testing problematic especially in infants
(Stetler et al 1997).
Indirect
ELISA is a quantitative method that involves the detection of serum antibodies
to HIV infection. Samples containing antibodies are added to a microtiter plate
with an antigen coating. The antigen coating is a solid support and the
reaction time is around 30 minutes at a temperature of about 37̊- 40̊ C. Upon
reaction unbound components are washed away and the antibodies attached to the
antigen on the solid surface are detected via the addition of an enzyme
containing a secondary anti-isomer type antibody that is capable of binding to
the antibody within the sample. A substrate for the enzyme is added and a
coloured product is formed and is analysed using a spectrophotometric plate
reader which measures the absorbance of the wells and OD values are produced.
The sensitivity of this test may be increased with the use of polyvalent
conjugates such as antibody-G and antibody-M and configurations of sandwich
ELISA. Patients infected with HIV will contain an assay with the recombinant
envelope and the core proteins of HIV which are absorbed as solid-phase
antigens to micrtotiter wells. Individuals with the infection would produce
antibodies to epitopes present within the viral protein which indirect ELISA is
able to indentify (Dessie et al 2008).
Competitive
ELSIA is an incubation method used to measure the content of antigen and is
similar to indirect ELISA and therefore may be used to detect certain sub-types
of HIV-1, where the HIV antibody competes with the enzyme bound antibody. The
protocol involves an initial step of incubation of antibodies in solution with
a sample of antigens. The antigen-antibody sample is then added to a microtiter
well coated with an antigen. As the amounts of antigen increases the amount of
free antibody decreases. Upon the addition of a conjugated-enzyme containing
secondary iso-type antibody specific for the iso-types, PAs may be used to
detect the quantity of primary antibody-antigen within the well. However, the
absorbance becomes low as the original antigen concentration increases within
the sample thus development of colour becomes inversely proportional to
specific HIV antibody concentration (WHO, 2002).
A
modified method using ELISA is the use of chemiluminescence which is convenient
luxogenic substrate that results in a 10 fold increase in the detection limit
during certain chemical reactions providing high sensitivity to measure
absorbance. The technique involves a further increase in detection by the
addition of an enhancing agent resulting in a 200 fold increase in the
detection limit. Furthermore, under ideal conditions the detection limit may
stretch up to 5 x 10-18 MOLES of
target antigens. Another modification of ELISA is the ELISPOT assay, which
allows the quantitative determination of the number of cells in a population
that is antibody specific for a specific antigen. This approach uses plates
that are coated with capture antigens which are recognised by the capture
antibodies specific for the antigen that is consistent with the assay. This
step is followed by the addition of a suspension of cells of the investigative
population into the coated plates and incubation. Upon incubation, the cells
settle on the surface of the plate followed by the reaction between secreted
and capture molecules producing a ring of antibody-antigen complexes around
each investigative cell. Upon the washing of the plates, an enzyme-linked
antibody specific for the secreted antigen or specific for the antibody is
added. Following binding between antigens and antibodies, a subsequent assay is
developed via the addition of chromogenic or chemilumenescence components
revealing the positioning of cells producing antibodies or antigens. This form
of ELISA is used amongst certain subjects suspected of HIV-1/2 (WHO, 2002).
The
protocols of the sub-types of ELISA are similar to one another when determining
the presence or absence of HIV-1/2. However, there are advantages and
disadvantages to the technique. Disadvantages of ELISA include; the hampering
of the analysis of peptide specific antibodies caused by the difficulties using
immobilized synthetic antigens on solid support which may vary with the type of
microplate and the composition of amino acid sequences within a peptide.
Currently, procedures are being designed to improve the coating of synthetic
peptides on solid phases via simple and rapid techniques based on the
irradiation of microplates with the use of UV light before the use of ELISA
techniques (Lequin, 2005). However,
pre-treatment is shown to improve the specific signal and minimise
dose-dependent response. Advantages of ELISA include the use of the method as
diagnostic tool amongst infants less than 18 months of age thus, allowing the
rapid anti-retroviral treatment to preserve the infant’s immune system
(Lujan-Zilbermann et al 2009). ELISA techniques are relatively inexpensive, has
the ability to adapt to large scale screening, less probability of false
negative results and high reproducibility (Winkler, 2005). Hence antibody
testing using ELISA is a favourable and reliable technique in the
identification of HIV.
