HIV SPECIFIC MONOCLONAL ANTIBODY PRODUCTION
Introduction
Antibodies belong to a group of proteins from the immune system called immunoglobulins. Each antibody has a Y-shaped structure made up of two heavy-chain and two light-chain polypeptides (Alzari et al., 1988, p. 558). Antibodies get their specificity from the highly-varied region at the tip of the Y-shaped structure (Burton & Woof, 1992, p. 46). This amino-acid sequence in this variable region consists of between 110 and 130 amino acids. The structure of antibodies makes them bind to specific antigens with a complementary binding site (Davies & Metzger, 1983, p. 94). Antibodies can be classified as monoclonal or polyclonal based on their binding ability. Monoclonal antibodies are generated from a single B-lymphocyte and can only bind to one specific epitope (Harris et al., 1992, p. 372). Conversely, polyclonal antibodies have several specificities and can bind to different epitopes because they are generated from different B-cell lines.
This diagram showing the structure of an antibody. There are two light chains, two heavy chains, a constant portion and a variable portion.
Monoclonal antibodies are produced artificially in large quantities from a cell type called a hybridoma, which has the potential to produce antibodies indefinitely (Kohler, 1986, p. 1284). These antibodies are able to recognize and bind to specific epitopes on antigens thus offering protection against disease organisms. Monoclonal antibodies work by targeting various proteins that facilitate cell activity. These include receptors and other surface proteins on normal and cancerous cells (Schmitz et al., 2000, p. 108). Due to their specificity, monoclonal antibodies are coupled with cytotoxic agents and targeted at cancer cells. This kills the cancer cells only while leaving out normal cells. Monoclonal antibodies are therefore more effective than conventional drugs, which target both normal and cancerous cells leading to harsh side effects (Lonberg & Huszar, 1995, p. 367). When there are high amounts of monoclonal antibody in the blodd, it means an abnormal protein has been detected. This abnormal protein can be identified through protein electrophoresis, a form of screening blood test.
Hybridomas are produced through the fusion of B-cells and myeloma tumor cells. The resultant hybrid cells are able to multiply rapidly and indefinitely since they are essentially cancer cells, and in the process they produce large amounts of antibodies (Mestecky & McGhee, 1987, p. 154). The newly formed hybridoma cells are selected using HAT medium (hypoxanthine-aminopterin-thymidine), which helps in eliminating unwanted types of myeloma cells. Hypoxanthine and thymidine are intermediate products of DNA synthesis while aminopterin is a powerful folate inhibitor drug. The combination of the three substances facilitates the artificial selection of cells that contain enzymes HGPRT and thymidine kinase (TK), which are needed for cell division and DNA sysnthesis (Ridzon et al, 1997, p. 923). The desired primary hybridoma cells are then cloned through limited dilution.
HIV is type of lentivirus that attacks the immune systems of human beings, weakening the body and leaving it exposed to a host of other diseases. The virus is spread through contact with the body fluids of infected persons. It is commonly transmitted though sex, contact with infected blood, or from mother-to-child (Van de Perre et al., 1991, p. 596). HIV virus infects cells of the human immune system, specifically CD4+ T cells. When the numbers of CD4+ cells are diminished to a critical level, the body loses the protection of cell-mediated immunity and eventually AIDS sets in. Screening tests for HIV used in clinics target HIV antibodies in the blood of an infected person. These are often rapid antibody tests that produce results in less than 30 minutes. There are also antibody-antigen tests that take several days.
Aim: The aim of this experiment is to produce HIV-specific monoclonal antibodies from hybridomas produced in the laboratory.
METHODOLOGY
The first step involves the production of hybridomas through the fusion of myelomas and B cells from a mouse infected with HIV virus. Once the B cells have been drawn from the spleen of the mouse, they are fused with myeloma cells that lack the HGPRT gene (MacDonald et al, 1989, p. 621). The fused cells are then incubated in the HAT medium. This causes unfused D cells and unfused B cells to die, leaving only B-cell myeloma hybrid cells to survive.
The next step involves dilution of the incubated medium into multiwall plates so that each well contains only one well. The hybridoma colony is established and continues to grow and produce antibodies in culture medium. A rapid screen process is used to identify and select only those hybridomas that produce the desired HIV-specific antibodies (Milstein, 1986, p. 1264). For this purpose, a dot blot technique was used.
After selection, the positive hybridomas are grown in larger tissue culture flasks to facilitate the multiplication and well-being of the cells. The antibody is derived from the culture supernatant and has to be kept at 20 degrees Celsius or below and also has to undergo a final purification. The two common techniques for purifying these antibodies are: (i) ion-exchange chromatography, and (ii) antigen affinity chromatography.
RESULTS
The dot blot test produced a positive reaction which presents as a coloured dot against the white filter background as seen in Figure 1 below. A clear visible spot was obtained using just a small drop of the sample, with a spot diameter of >0.5mm. The use of nitrocellulose enables the results of the reaction to be viewed against a white background for clarity. This provides better discriminatory power when needed as compared to other methods like the microtiter plate. It becomes easier to distinguish between positive reactions and false positives.
Figure 1: blot spot test results
The result for the western strip method used for detecting HIV antibody was also positive as can be seen in the Figure 2 below. HIV antibodies bind to the proteins in the test area resulting in the appearance of a red line. A positive result is seen when two red lines appear – one in the test area and another in the control area. A negative result is seen when there is only one line seen in the control area. A sample is considered positive if the line has a clear red colour as seen in the picture.
Figure 2: strip test results
DISCUSSION
For figure 1, the dot blot represents a positive result for the presence of HIV antibodies. The type of profile produced is used to determine whether an individual is considered positive for HIV antibodies. CDC guidelines require reactivity to at least two of the following antigens for it to be a positive classification – p24, gp 41 or gp 120/160. A negative result is represented by the absence of all bands. Results can also be declared as negative if there is only a very weak p17 band. Sometimes there can be reactivity to one or more antigens but does not fulfil the criteria for positivity. These are considered indeterminate results. Sera from healthy non-infected individuals can show some reactivity to one or more antigens when tested using this method (Vlasek & Ionescu, 2008, p. 468). This occurs in up to 15 percent of non-infected individuals. In most of these indeterminate results, only weak reactions occur involving the Gag proteins. Other patterns may be seen but less frequently. Any outcome that does not qualify clearly as positive or negative is considered indeterminate.
Some of the individuals whose screening produces indeterminate results with western blot method sero-convert later, such that their p24 and p55 profile can be indicative of early infection (Fiebig et al., 2003, p. 1884). On the other hand, some individuals never sero-convert and maintain the same profile for years, meaning that they are not infected. In fact this occurs in most cases; therefore an indeterminate result with western blot cannot be used to predict early infection. Individuals who show indeterminate results are supposed to be retested after several months, even though they can sero-convert and be detected much earlier (Burke et al, 1988, p. 963). Whenever possible, the retesting should be done in parallel with a reassay of the original sample under the same conditions for direct comparison.
The HIV virus carries many unique proteins on its surface and inside. HIV antibodies are directed at these unique proteins but unfortunately do not eliminate the virus. Nevertheless, the presence of HIV antibodies is used as a marker to determine those infected with HIV. In the western strip test (figure 2), a person’s serum is made to react with virus proteins on the strip. If the serum is infected with HIV, it will contain antibodies that will bind the HIV proteins. This reaction is represented in the form of a red line in the test area. There is a small chance that a false positive result can be obtained therefore a second testing is done for cases that are initially positive. The western blot test is commonly used a second time when the strip test produces a positive result. The HIV proteins are separated based on electric charge and size. A positive test will produce a series of bands meaning the person’s antibodies are binding to specific HIV virus proteins. If the serum is negative for HIV, there will be no separate bands produced on the result paper.
Other methods have been developed for testing HIV in a more accurate and conclusive manner (Armstrong & Taege, 2007, p. 298). Polymerase chain reaction (PCR) test is used to detect HIV RNA in human blood. This is based on the fact that HIV RNA is different from all human DNA. PCR tests are commonly used for neonatal screening of HIV-positive mothers (Farzadegan et al., 1993, p. 340). There is a chance of maternal antibodies crossing the placenta and entering the newborn. This would result in a positive antibody test even when the infant is not infected with HIV (Beck et al, 2010, p. 349). PCR also detects HIV within the first four weeks following exposure, something not possible with antibody tests.
There is also the HIV combination test which detects not only HIV antibodies but also a protein called p24. This protein is found in the core of the HIV virus. It is important to detect this protein because antibodies take weeks to form against the HIV virus even though the virus and the p24 protein are already in the blood (Kleinman et al., 1998, p. 280). Combination testing enables HIV diagnosis to be made within the first week of infection. With this test, a light is emitted and registered on a detector whenever either the antibody or p24 protein is present in the blood. Tests for detecting the p24 protein alone are known as antigen tests and they target monoclonal antibodies against the capsid protein (Mas et al., 1997, p. 67). An enzyme linked to the monoclonal antibodies binding the p24 protein will cause a colour change when p24 protein is detected in the sample.
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