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HIV SPECIFIC MONOCLONAL ANTIBODY PRODUCTION
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.
References
Alzari, P. M., M.-B. Lascombe, and R. J. Poljak. (1988). Three-dimensional structure of antibodies. Annual Review of Immunology. 6, pp. 555–580.
Armstrong W. S., Taege A. J. (2007). HIV screening for all: the new standard of care. Clinical Journal of Medicine, 74 (4), pp. 297–301.
Beck A., Wurch T., Bailly C., Corvaia, N. ( 2010). Strategies and challenges for the next generation of therapeutic antibodies. National Review of Immunoogyl. 10 (5), p. 345–52.
Burke D., Brundage J, Redfield R, Damato J, Schable C, Putman P, Visintine R, Kim H (1988). Measurement of the false positive rate in a screening program for human immunodeficiency virus infections. New England Journal of Medicine 319 (15): 961–4.
Burton, D. R., and J. M. Woof. (1992). Human antibody effector function. Advanced. Immunology. 51:1–84.
Davies, D. R., and H. Metzger. (1983). Structural basis of antibody function. Annual Review of Immunology. 1:87–117.
Farzadegan H, Vlahov D, Solomon L, Muñoz A, Astemborski J, Taylor E, Burnley A, Nelson K (1993). Detection of human immunodeficiency virus type 1 infection by polymerase chain reaction in a cohort of seronegative intravenous drug users. Journal of Infectious Diseases. 168 (2): 327–31.
Fiebig, E. W.; Wright, D. J.; Rawal, B. D.; Garrett, P. E.; Schumacher, R. T.; Peddada, L.; Heldebrant, C.; Smith, R.; Conrad, A.; Kleinman, S. H.; Busch, M. P. (2003). Dynamics of HIV viremia and antibody seroconversion in plasma donors: Implications for diagnosis and staging of primary HIV infection. AIDS (London, England) 17 (13): 1871–1879.
Harris, L. J., S. B. Larson, K. W. Hasel, J. Day, A. Greenwood, and A. McPherston. (1992). The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 360, p. 369–372.
Kleinman S, Busch M, Hall L, Thomson R, Glynn S, Gallahan D, Ownby H, Williams A (1998). False-positive HIV-1 test results in a low-risk screening setting of voluntary blood donation. Retrovirus Epidemiology Donor Study. JAMA 280
Kohler, G. (1986). Derivation and diversification of monoclonal antibodies. Science 233:1281–1286.
Lonberg N., and Huszar D (1995). Human antibodies from transgenic mice. International Review of Immunology. 13 (1): 65–93.
MacDonald K, Jackson J, Bowman R, Polesky H, Rhame F, Balfour H, Osterholm M (1989). Performance characteristics of serologic tests for human immunodeficiency virus type 1 (HIV-1) antibody among Minnesota blood donors. Public health and clinical implications. Ann Intern Med 110 (8): 617–21.
Mas, A.; Soriano, V.; Gutiérrez, M.; Fumanal, F.; Alonso, A.; González-Lahoz, J. (1997). “Reliability of a new recombinant immunoblot assay (RIBA HIV-1/HIV-2 SIA) as a supplemental (confirmatory) test for HIV-1 and HIV-2 infections”. Transfusion science 18 (1): 63–69.
Mestecky, J., and J. R. McGhee. (1987). Immunoglobulin A (IgA): Molecular and cellular interactions involved in IgA biosynthesis and immune response. Advanced Immunology. 40:153–246.
Milstein, C. (1986). From antibody structure to immunological diversification of immune response. Science 231:1261–1268.
Ridzon R, Gallagher K, Ciesielski C et al. (1997). “Simultaneous transmission of human immunodeficiency virus and hepatitis C virus from a needle-stick injury”. N Engl J Med 336 (13): 919–22.
Schmitz U, Versmold A, Kaufmann P, Frank HG (2000). “Phage display: a molecular tool for the generation of antibodies—a review”. Placenta 21 (Suppl A): S106–S112.
Van de Perre P, Simonon A, Msellati P, Hitimana D, Vaira D, Bazubagira A, Van Goethem C, Stevens A, Karita E, Sondag-Thull D (1991). “Postnatal transmission of human immunodeficiency virus type 1 from mother to infant. A prospective cohort study in Kigali, Rwanda”. N Engl J Med 325 (9): 593–8.
Vlasek J, and Ionescu R (2008). “Hetergeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods”. Current Pharmaceutical Biotechnology 9 (6): 468–481.
Ethics of Ambiguity
Ethics of Ambiguity
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Introduction
The importance of philosophy in the contemporary human society cannot be gainsaid as far as critical thinking is concerned. Indeed, this is the fundamental use of philosophy as a discipline. Needless to say, the human society has seen numerous philosophers with varying degrees of popularity, as well as different philosophies. Philosophers have, in varied instances, been striving to explain the essence of human existence. This was the case for Simone de Beauvoir, a French existentialist philosopher who sought to create an enhanced understanding or comprehension of what individuals are as human beings. She stated that the existence of individuals was ambiguous as it is never clear exactly what individuals are as human beings, in which case they must face their problems via life itself as they go through it (Velasquez, 2011). De Beauvoir outlined varied attempts at resolving the ambiguity that clouds an individual’s life or existence, one of which is seriousness or “the serious man”.
The comprehension of seriousness necessitates that individuals examine the manner in which they were brought up or conditioned. Kids start their lives with a considerable level of seriousness, which underlines the fact that they are born into worlds that they stand no chance of changing (Velasquez, 2011). This means that they have no option but to submit to the world, irrespective of how much they hate it. For instance, when children are told that cheating or lying is wrong, they have to accept that notion as an absolute or supreme moral law. He would not have to question the basis upon which such a notion is made or even why cheating or lying is wrong.
However, this worldview develops some contentions or cracks once an individual gets into adolescence. Children, as teenagers, usually notice the contradictions that are rife in their parents and other adults’ lives, as well as the world in its entirety. For instance, they may realize that their parents lie to each other or even to other kids, even after telling them that cheating or lying is wrong. This underlines the fact that a close examination of the life that the child lives would reveal that their own customs and values emanate from adults. It is, in essence, at this point that the individual would realize or achieve his freedom. Indeed, they realize that they too, will have to become adults and make their own choices. However, this comes as a mixed blessing as the world is not outside the control of the child, rather, he has to make a contribution to it and become successful.
The child may reach a point in which he would be wary of his or her freedom and afraid of the fact that he would have to interact with the world and adhere to the laid down rules and regulations. He will, undoubtedly, remain nostalgic of the time when he did not have to make a choice, a time when everything would be laid out before him (Velasquez, 2011). Beauvoir states that the man, instead, would take refuge in the serious world that he, at one time, knew as a child. This time, however, the individual would pretend that the values that he holds or espouses are real, objective, as well as independent of other human beings, while, in fact, they have been created by other men. In this case, the man, unlike the child would be making a choice even in instances where he does not think that he is (Velasquez, 2011). This means that the serious person would be claiming to take refuge in habits and values that do not originate from human beings, while, in fact, there exists no such values and rules. He would be lying to himself if he thought that the values are not conditioned.
In some cases, the serious man would join a group of people such as Communists or Christians, identifications that would, eventually give him some rights. This may inspire a belief in the man as to the objectiveness of the rights, which is worn as he would have chosen them. Beauvoir notes that the serious attitude would have the capacity to easily result in fanaticism, where other people’s freedoms would be entirely ignored for some ideal that is supposedly objective (Velasquez, 2011). The serious human being would have to sacrifice himself or herself to a particular ideal that he would have chosen.
Beauvoir states that the world creates by oppression is plagued by seriousness (Velasquez, 2011). This affirms the oppressive order and christens is a “natural situation”, a world that an individual would be incapable of changing, and against which it would be impossible and hopeless for an individual to successfully revolt. In addition, it would be impossible for an individual to know the joy that comes with the destiny pertaining to human existence that is entangled in the serious world, or even the world pertaining to the child (Velasquez, 2011). Just as the mythical first individuals had to exchange their paradise for the pains and pleasures that come with knowledge, existentialists viewed the human condition as incorporating a brokerage of the pleasures that come with child’s play for the fretfulness pertaining to subjectivity, as well as its joyful possibilities. Beauvoir states that human beings can only escape from the serious world through a revolution or a thoroughgoing rebellion. Human beings cannot just come up with modest alterations to such a world, rather the oppressed would fulfill and achieve their freedom as human beings only through revolting, especially considering that the essential features pertaining to the situation against which they are rebelling precisely revolves around prohibiting them from achieving any desirable development.
As much as revolutions would be essentially difficult and troublesome to individuals, Beauvoir justifies them on the basis that this is the only way that an individual would be free and have the capacity to chart his own destiny. Indeed, it goes without saying that individuals live by rules that are created by other human beings living in the same world before them. However, as much as the rules that they make are time-tested and could, in fact, be extremely efficient in ensuring stability and peace, different circumstances will, undoubtedly, necessitate the crafting of different rules and regulations. Indeed, rules and regulations often change, become irrelevant and inapplicable as individuals get into different situations (Velasquez, 2011). It is, undoubtedly, extremely enslaving for individuals to be forced to live under rules whose applicability has been compromised by the sands of time, or whose relevance is compromised by the prevailing circumstances. However, piecemeal changes would be insufficient in eliminating such oppression, in which case it would be imperative that a widespread revolution is undertaken to liberate the individuals.
References
Velasquez, M. G. (2011). Philosophy: A text with readings. Boston, MA: Wadsworth/Cengage Learning.
Resistance to Apartheid in South Africa
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Resistance to Apartheid in South Africa
Apartheid in South Africa was one of the major defining events and factors in the history of the country as it made a lasting impact on its independence. It also defined the place of the whites and the place of the blacks in the country. The citizens of South Africa at that time were of great concern towards how they were treated by the whites and therefore had to develop ways of combating this. Due to the different effects of apartheid, they made decisions which to some extent were considered crude and therefore had to be used. However other people especially nelson Mandela opted for peaceful ways of making sure that there was no destruction which took place and therefore he led a group of South Africans who made sure that the resistance was a peaceful one and not one which led to the shedding of blood as some citizens and individuals suggested (Clark,20).
The effects of apartheid were seen in that the wealthy class was all whites while the poor class was all blacks and therefore this infuriated the blacks and it was the time for them to come up with a revolution that made the whites realize that what they were doing was wrong. It is also this resistance that came up that made the country to be sane and balanced in terms of the end to the apartheid practice whereby the blacks were discriminated against in line with their color of skin.
The ANC youth league emerged in 1940 after its president who was called Albert Luthuli made a rigorous attempt to revive it and make sure that the black folk could be respected by the whites(Clark et.al, 59). He was assisted by his fellow leaders and friends who were; Oliver Tambo, Nelson Mandela, water Sisulu. These four individuals were a great source of change for the South African people. Their unity and power to change the country led many into positivity and therefore there was hope that it will be possible for the whites to be equal and treated the same as blacks. The population hoped that with their assistance the whites could stop treating the blacks as evil people but rather begin treating them as equal human beings who deserve respect. In addition to the ANC youth league, the Indians who were living in South Africa also formed their movement which they called the South African Indian congress which voiced their lamentations and complaints about the way they were treated negatively by the whites.
These two groups organized a defiance campaign in 1952 and it was one of the most successful times for the fight against apartheid rule and the superiority with which the whites treated themselves with. This can be said to have been the birth of the congress of the people which happened 3 years later after the defiance campaign. All these efforts were important steps in the fight against the apartheid rule by the blacks. It was during this congress of the people when the freedom charter was adopted and therefore they came up with the statement that the whole country of South Africa belongs to all the people who live in it and not only to the whites. It also stated that no government can be justified to bring about rules and the manner of leadership they want as long as it is not based upon the people. This sounded closer to the democratic tenet that freedom belongs to the people and therefore the country belongs to the people and they are the ones who make decisions on how they want to be ruled through the different ways in which they come up with.,
At this time of the demonstrations and resistance, the government was always involved in disrupting the peace and the unity of the protestors by using force and making them feel unable to continue with the protest and the resistance. This was true for the congress of the people whereby the government broke in and arrested around 150 individuals. This was supposed to scare them however it never made the campaign die. In 1968 the government of South Africa came up with more ways to block the blacks and other people of color from resistance by creating laws to ban such kinds of activities. This was done when the prohibition of the political interference act was passed. This to some extent made the government win against its citizens.
However, with the rising need for liberation, Robert Sobukwe led a group of individuals to form a different movement from the ANC to address some of the issues which they felt were not being addressed. Through his efforts, they founded the pan-Africanist congress. This movement focused solely on black liberation and they did not include other groups like the Indians. This they did because they believed the progress of the ANC was being undermined by its incorporation of other groups. However with all these efforts the police still never gave peace to the blacks and they still pushed ahead to start a fire at Sharpeville which harmed many people and killed others. This was one of the cruelest ways which were adopted to make the blacks and people of color silent about what they were going through (Roger, 25).
Even though the government was successful in maintaining apartheid under control for almost a decade it was not completely successful since after the decade it came back with even more power and more resistance which proved difficult to control and hold. In 1973 the black trade unions organized a series of strikes and this was not only for apartheid and racism but for better working wages and better working conditions. However, on the 26th of July 1976 thousands of children in the Soweto slums demonstrated against the several policies of the government and one of them was the insistence of the government for them to be taught in Afrikaans rather than English. This was one of the greatest events in the history of southern Africa and it aroused international concerns which had a great impact tot eh country. The struggle continued and it was a difficult time for the black people until the time Nelson Mandela was president when everything came to a standstill and it was possible for the blocks to feel at peace with their land.
Therefore the period of resistance was a difficult one for all kinds of people in South Africa. Even though it affected blacks more than the whites it still affected the whites like the hate which the blacks and other people of color harbored and therefore it was possible for any white person to be done badly by any black group of individuals. The effects of apartheid and racial discrimination range from personal, communal, and national ones. As discussed above the main methods of resistance were organizing movements together and using them to demonstrate and to bring a collective voice together to air their issues which were never listened to. This is a very crucial method of resistance because there is always power in the voice of large numbers (Tom, 726).
Therefore in conclusion we can say that the resistance was successful after a long time of rejection and being pissed off by the government. The rule of nelson Mandela was a very positive thing for the blacks and this acted as one of the best ways of liberation from how they were doing their things.
Works cited
Clark, Nancy L., and William H. Worger. South Africa: The rise and fall of apartheid. Routledge, 2016.History.com Editors. “Apartheid.” HISTORY, A&E Television Networks, 7 Oct. 2010, www.history.com/topics/africa/apartheid.Lodge, Tom. “The rise and fall of Apartheid.” Canadian Journal of political science 45.3 (2012): 726.
Southall, Roger. “The ANC & black capitalism in South Africa.” Review of African Political Economy 31.100 (2004): 313-328.