Enzyme Immunoassay Principle

Enzyme Immunoassay Principle ENZYME IMMUNOASSAYS (EIA) Enzyme immunoassay (EIA) INTRODUCTION Enzyme immunoassay (EIA) or enzyme linked immunosorbant assay (ELISA) is a powerful technique used for detecting and quantifying antigens and antibodies in clinical samples. It is widely used in clinical laboratories for diagnoses, prognoses and for monitoring immune responses. The aim of this essay is to summarise the principle of this technique and to outline the main types of enzyme linked immunosorbant assay (ELISA) and its application in clinical laboratories. Subsequently, it will discuss the application of this system in the haematology field. PRINCIPLES The general principle of this technique is based on the binding of conjugated enzyme molecule with specific antibodies to detect and quantify the presence of either antigens or antibodies in the test sample. This is followed by adding appropriate colourless substrate which catalyses the interaction complex to produce a visible coloured product. There are many chromogenic substrates used in ELISA technique but the most common are alkaline phosphatase (AP) and horseradish peroxidase (HRP). The end product can be determined by using spectrophotometer and the intensity of colour is directly proportional to the presence of either antigens or antibodies in the test samples. TYPES There are several types of ELISA which include: indirect, sandwich, competitive, chemiluminescence and elispot assay. Indirect ELISA can be used to detect the antibodies that are present in patients serum. With this method, patients serum containing antibodies is added to the microtitre wells which are coated with the antigen and the intensity of the end colour product is directly proportional to the amount of antibodies that are present in the patients sample. (KUBY) Figure1. Illustrate the different types of ELISA to detect either antigen or antibody. The second type of enzyme linked immunosorbant assay is called sandwich ELISA, in which the target antigen that is to be detected must be located between two antibodies. The antigen of interest that is present in the clinical sample is added to a microtitre wells which is coated with the antibody and the end product is determined by spectrophotometer to measure the amount of antigen in the clinical sample.(DIAGNOSTIC MICRO. BOOK) Figure1. The development of biotechnology has become widespread and one example of its progress is the modification of ELISA by using chemiluminescence substrate rather than chromogenic substrate to increase the sensitivity of the reaction. With this method, chemilumescence (CL) substrate and some enhancing agents, which have the ability to bind an antibody or antigen in the reaction, are used to create light emissions which can be measured by specific spectrometry. This approach has been used to diagnose several types of tumour disease, and quantify numerous compounds at low concentration (Baeyens et al. 1998). Another example of ELISA modification is enzyme linked immunospot (ELISPOT) assay. It is a quantitative technique often used to detect cytokines that are secreted from single cell in response to the antigen. During the incubation period, the cytokines of interest is released and captured by immobilised anti-cytokines on the microtitre plate. After washing them and adding labelled anti-cytokines antibody and substrate, the end colour product displays as a footprint around each of the secreting cells, which can be estimated. This technique has great potential for monitoring immune responses (Cox, Ferrari and Janetzki 2006). In addition, sensitive ELISPOT assay has the ability to detect low level of secreted IFN-? compared with other IFN-? assays (Lion et al. 2009). APPLICATION There are many applications of enzyme immunoassays in clinical laboratories and the largest application is related to the detections of viruses. For example, the screening and diagnosis of hepatitis virus is routinely used in the lab. These include the detection of HBsAg and HBeAg by direct ELISA, anti-HBc antibody and anti-HBe antibody by competitive ELISA and anti-HBs antibody by indirect ELISA (Tsitsilonis et al. 2004).What is more, ELISA can detect HCV antibodies in serum samples as screening tests and the positive samples are conformed by molecular HCV RNA (Erensoy 2001). The beneficial effect of using an enzyme immunoassay in clinical hematology is that it has the ability to identify alloantibodies in patients who frequently receive platelet transfusions for therapeutic purposes. It has been demonstrated that ELISA is more sensitive to the detection of alloantibodies than other immunoassays, such as lymphocytotoxic test (LCT) and the platelet immunofluorescence test (PIFT), and it is performed in a short period of time (Levin et al. 2003). Furthermore, enzyme immunoassays are able to detect anti-platelet antibodies, platelet associated IgG (PAIgG) , HLA and circulating antibodies in patients with Idiopathic thrombocytopenic purpura (ITP) (Schiffer and Young 1983). There are several pieces of evidence which suggest that a ubiquitous herpes virus, in particular Epstein-Barr virus (EBV), is implicated in many types of lymphoma such as Hodgkins disease and Burkitts lymphoma, so the early detection of EBV antigen specific T cells is essential and therefore ELISPOT assay can be used to analyse T cell responses (Yang et al. 2000). Another of its feature is related to acquired von Willebrand syndrome, which is caused by autoantibodies against von Willebrand factor (vWF). This plays an essential role in hemostasis and is characterised by prolonged bleeding due to the defect in vWF activity. Detection of anti-vWF antibody can be performed by ELISA (Mohri 2003), and (Stewart et al. 1997) mention that competitive ELISA is a useful technique in the detection of anti-vWFA antibodies in a clinical samples. Despite the benefits of ELISA in the detection of HLA class I specific antibodies, it is less sensitive than flow cytometry, so before platelet transplantation, flow cytometry must be used (Worthington et al. 2001). Although chemiluminescence immunoassay has great sensitivity in detecting HBsAg in blood donor samples, it has less sensitivity than the polymerase chain reaction (PCR)(Sato et al. 2001), so to increase the safety of blood from HIV-1 , HCV, and HBV, the nucleic acid amplification technique (NAT) has been applied for the screening of blood donor samples (Weber, MÃ ¼hlbacher and Melchior 2005). CONCLUSION Enzyme Immunoassay is one of the most significant techniques to have been used in clinical laboratories to analyse the antigen-antibody reaction in a short period of time. It is sensitive to screening a large number of clinical samples and therefore has been applied in many clinical laboratories for diagnoses, prognoses and for monitoring immune responses. REFRENCES BAEYENS, W. R. G., et al. (1998). Chemiluminescence-based detection: Principles and analytical applications in flowing streams and in immunoassays. Journal of pharmaceutical and biomedical analysis, 17 (6-7), 941-953. COX, Josephine H., FERRARI, Guido and JANETZKI, Sylvia (2006). Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods, 38 (4), 274-282. ERENSOY, Selda (2001). Diagnosis of hepatitis C virus (HCV) infection and laboratory monitoring of its therapy. Journal of clinical virology, 21 (3), 271-281. LEVIN, M. D., et al. (2003). Screening for alloantibodies in the serum of patients receiving platelet transfusions: A comparison of the ELISA, lymphocytotoxicity, and the indirect immunofluorescence method. Transfusion, 43 (1), 72-77. LION, E., et al. (2009). Quantification of IFN-? produced by human purified NK cells following tumor cell stimulation: Comparison of three IFN-? assays. Journal of immunological methods, 350 (1-2), 89-96. MOHRI, H. (2003). Acquired von willebrand syndrome: Its pathophysiology, laboratory features and management. [online]. Journal of thrombosis and thrombolysis, 15 (3), 141-149. SATO, S., et al. (2001). Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion, 41 (9), 1107-1113. SCHIFFER, CA and YOUNG, V. (1983). Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA). [online]. Blood, 61 (2), 311. STEWART, M. W., et al. (1997). vWf inhibitor detection by competitive ELISA. Journal of immunological methods, 200 (1-2), 113-119. TSITSILONIS, Ourania E., et al. (2004). Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA). Journal of pharmaceutical and biomedical analysis, 34 (4), 811-822. WEBER, Bernard, MÃÅ"HLBACHER, Annelies and MELCHIOR, Walter (2005). Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing. Journal of clinical virology, 32 (1), 67-70. WORTHINGTON, J. E., et al. (2001). A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies. Human immunology, 62 (10), 1178-1184. YANG, J., et al. (2000). Application of the ELISPOT assay to the characterization of CD8(+) responses to epstein-barr virus antigens. Blood, 95 (1), 241-248.

Charles Dickens Essay

Thomas Hardy was a 19th century novelist and a 20th century poet. As a novelist, he was last of the great Victorian novelists such as William Thackeray, Charlotte and Emily Bronte, Charles Dickens and George Eliot. The last decade of the 19th century was dominated by Thomas Hardy. He wrote 14 novels and almost 900 poems. Hardy’s reputation as a novelist grew during the last decades of of his life and his poetry was relatively neglected. His novels share a pessimist view of the human condition and life. Pessimism is derived from the Latin word ‘Pessimism’ (worst). It is based on a belief that the world is the worst possible and that things are bad and tend to become worse. Thomas Hardy worked out a pessimist theory of his own according to which man is just a puppet in the hands of an inscrutable and malicious force which governs the world and seems to enjoy inflicting endless sufferings. The fact that Hardy resented being called a pessimist is no reason why should not be thus described. Hardy was the painter of darker side of life as it was no wonder if people charge him of ‘pessimist’. The opinion is both right and wrong in this context. In fact, there are some factors that compel us to believe him a pessimist. He was hyper-sensitive, his own life was tragic and gloomy. For a speculative soul, this world is a thorny field. Thomas Hardy captured the heartbeat of the rural English people against the looming backdrop of encroaching industrialism. His novels have a genuine, almost autobiographical feel because he used many personal experiences, acquaintances, settings and opinions in his fiction. Thomas Hardy’s pessimism also represents actual events. Relationships and social issues of Hardy’s life. Hardy’s fatalism and pessimism began to manifest itself in his early childhood, as he was the result of an unplanned pregnancy, and then later in his youth, when his family could not afford to fund a full education. As he grew into adulthood, Hardy began to feel acutely the line drawn between him and those of a higher class. His despairing relationships with two women, his cousin Tryphena Sparksand his first wife, Emma Lavinia Gifford, were the inspiration for the futile plotlines of his three novels. Because of the harsh society in which he lived, his lack of money, two unhappy relationships, and the failure of his last two novels to be accepted by his readers; (due to their anti-marriage, anti-social and anti-religious material), Thomas Hardy emerged as a pessimistic novelist and poet of the 19th century. Because of autobiographical touch in his novels, some of the Hardy’s heroes and even heroines adopt his personal qualities and go through some of the same life situations that he did. Additionally, many of the Hardy’s female characters are extremely similar to the lovers and friends he had throughout his life. Hardy personally felt the crushing pressure of the Victorian society, namely its rules and regulations concerning love and marriage. Hardy, and therefore, his characters often rebel against this society, making decisions that contradict the expectation of their society. Many times, Hardy felt that he was an outcast in his society, partly because his religious beliefs did not match up with the church’s, but also because his social thoughts and actions were more progressive than his counterparts. After experiencing years of disagreement with the England of the 1800 s and having a marked desire for progression, Hardy became understandably morose. His novels became increasingly dismal and pessimistic and it is for this fatalistic tone that he is greatly remembered. The Victorian age was an age of doubt, of contradictions and conflicts. This fact too shows its impact on the writing of Hardy. People were to live by the Bible but many took it in the strict sense and followed the literal words strictly we see in ‘Tess of the D’Urbervilles’ how Tess is treated unjustly by the society, which followed the law in words and not in spirit. The gloomy effect of his age plays an important role in his writings. Doubts, despair, disbelief, frustration, industrial revolution, disintegration of old social and economic structure, Darwin’s theory of evolution were gthe chief characteristics of that age. All these factors probe deep into his writings, and heighten the somber , melancholic and tragic vision. His pessimism is also the outcome of the impressions that he receives from villager’s life. There were plenty of tragedies in the life of the poverty stricken Wessen folk, as shown in this novel ‘Tess of D’Urbervilles’. Hardy’s philosophy of the human condition is determined by his natural temper and disposition, He says: â€Å"A man’s Philosophy of life is an instinctive, temperamental matter. † Hardy, practically, excludes from his writings the sense of splendour and beauty of human life completely. Tess’ life is totally devoid of even a single moment of happiness.

Criminal Profiling Essay

Criminal profiling involves analyzing the scene of a crime and then use the information gathered from the analysis to determine correctly the identity of a perpetrator. This method of identifying perpetrators is very useful though it does not assist an investigator in getting the name of the perpetrator it helps narrow down and thus makes it easier to identify a criminal. (Dennis, 2006) Some of the pertinent issues that criminal profiling helps in bringing out include but not limited to the sex of the perpetrator, the background of a person in terms of ethnic community, age and personality. This profile is then used to single out a criminal among several people who appear to match with some of the features identified in the profile. One may wonder just how does criminal profiling work? Normally experts examine the manner in which a particular crime is committed. This is commonly referred to as the method of operations. Usually the experts will take the identity of the victims and then examine what the suspected criminals have in common especially in terms of the likely weapon that was used to commit the particular crime in question. The other issue that the experts look at is the brutality or hostility that was used during the commission of the crime in question by the perpetrator. Existence of torture or any form of hostility is yet another trait that experts look for when using the criminal profiling method. One student in an American University during a lecture asked her professor why he believed that the criminal profiling method actually works in identifying a perpetrator in a criminal act. The professor gave a precise answer and stated that thorough research has been carried out over the years and there have been conducted some interviews even with convicts who have helped prove the workability of this method. There are two main ways that are used to come up a criminal profile. One of the methods is one that is commonly used by the Federal Bureau of Investigation known as criminal scene analysis. This method involves a number of steps which include profiling inputs. This basically involves gathering evidence in terms of anything that was found in the scene of the crime. The second step usually is to put the evidence together and try to see if there is any common aspect in the crimes committed by the suspects. Crime assessment and investigation follows and it is after that some apprehensions can be made based on the investigations. The second method is the behavioral evidence analysis. This method was developed by Brent Turvey who argued that when a crime is being investigated there are a lot of discrepancies that rise from all the gatherings that are made by the investigative team. The first step is forensic analysis which involves analyzing physical evidenced gathered which may involve taking photographs and videos of the crime scene and having interviews with some of the victims of the crime who may be alive. Victimology is the nest step and involves creating a profile for a victim which in turn helps identify the criminal responsible. The third step is examining keenly the features and characteristics of the scene of crime as this is said to give a leading clue on who the suspect could be. The last step is combining the first three steps discussed and it is this combination that helps the investigators come up with a criminal profile. According to Turvey a crime ought to be analyzed in four main steps to ensure that the information gathered by the investigative team does not bear any discrepancies. Criminal profiling has in several instances been criticized as a source that lacks credibility on the basis that it is based on speculations. However experts state that according to research that has been conducted for several years now, criminal profiling has a considerably high level of credibility. Profiling involves a number of phases. The initial phase in every criminal profiling is known as antecedent which involves attempting to figure out the plan or the fantasy that the perpetrator of a crime had prior to the commitment of the crime. Basically this phase of criminal profiling involves an attempt to unearth the motive behind the commission of a crime by the perpetrator. The second phase is known as method and manner and involves examining the target group that the perpetrator was aiming at in the crime committed and more so the manner in which or better still the method used to commit the crime in question. Brent, 2002) The third phase in criminal profiling is known as body disposal and is common in murder cases. This is where experts attempt to examine where the scene of crime is the same with the place where the body was dumped or whether the crime was committed at a particular place and then the body dumped in yet another place thus introducing the concept of multiple. (Davis, 2007) Post offense behavior is the final or the last phase in criminal profiling and it is quite tricky. Post offense behavior involves investigating behavior of criminals especially after commission of crimes. Normally some criminals try to hide their identity by pretending to be part of the investigating team. Experts must therefore use their expert knowledge to single out criminals who pretend to be assisting in the investigation process yet they are the suspects. Having examined in great depths what criminal profiling is all about, it may be of paramount importance to address the issue of whether criminal profiling has managed to shape up chance. Basically by making this analysis we are able to know whether criminal; profiling does any play any vital role in helping to identify criminals in a given crime in question. In simpler terms using criminal profiling as a method of identifying criminals what are the chances of identifying the suspect responsible in the criminal act at hand. In the beginning of the twentieth century, criminal law took a new twist with criminal profiling slowly shaping chance. Majorly what changed during the twentieth century was the inference in terms of probability. During this century actuarial methods changed. One thing to note that it is not the risk analysis and actuarial thinking that actually changed but the inferences in terms of probability. The actuarial methods got more refined so that using the criminal profiling method one can be sure that the identity of a particular perpetrator is correct. In conclusion therefore it is correct to state that over the year and more so after the twentieth century, criminal profiling is slowly gaining acceptance as reliable method of correctly identifying perpetrators of crimes. This is because it has been studied and proved that it shapes chance so that the probability of a person to be the suspect of a crime in question having been pointed out by the criminal profiling method is very high.

The Worst Hard Time Essay - 1882 Words

One theory in Jared Diamond s Collapse is that soil degradation and erosion leads to insufficient agriculture and a society s demise. In Timothy Egan s The Worst Hard Time, he sets forth in specific and excruciating detail exactly what Diamond outlines in Collapse. Only Egan s book isn t theoretical. It isn t a survey of what s happened in other countries. It s about the Dust Bowl in the 1930s. It s about what happens, right here in the heart of America, when the land is misused, mistreated, and turns on those who depend on it. Centered in the panhandle of Oklahoma and extending south into the panhandle of Texas, north into half of Kansas and a quadrant of Colorado, the Dust Bowl despoiled 100 million acres. For thousands of years this†¦show more content†¦As long as the weave of grass was stitched to the land, the prairie would flourish in dry years and wet. The grass could look brown and dead, but beneath the surface, the roots held the soil in place; it was alive and dormant. The short grass, buffalo and blue grama, had evolved as the perfect fit for the sandy loam of the arid zone. It could hold moisture a foot or more below ground level even during summer droughts, when hot winds robbed the surface of all water-bearing life. In turn, the grass nurtured pin-tailed grouse, prairie chickens, cranes, jackrabbits, snakes, and other creatures that got their water from foraging on the native turf.Through the driest years, the web of life held. When a farmer tore out the sod and then walked away, leav ing the land naked, however, that barren patch posed a threat to neighbors. It could not revert to grass, because the roots were gone. It was empty, dead, and transient. By the fall of 1930, the first dust storm rolled out of Kansas, through Oklahoma, and into the panhandle ofTexas. . . . [I]t rolled, like a mobile hill of crud, and it was black. When it tumbled through, it carried static electricity, enough to short out a car. And it hurt, like a swipe of coarse-grained sandpaper on the face. Yet the 1931 harvest was the largest ever, more than 250 million bushels, with 33 million acres plowed up in the southern plains, and theShow MoreRelatedthe worst hard time1139 Words   |  5 Pageshorizon today? Drawing on more contemporary examples of environmental disasters or concerns, write a paper that explores how this debate continues to be timely or that takes a stand on this debate. 2. According to the Houston Chronicle, â€Å"The Worst Hard Time documents how government and business with the best of intentions can facilitate the destruction of an entire region.† Explain how this is true with regard to the Dust Bowl, and then extend your analysis to include the relevance of this statementRead MoreThe Worst Hard Times Essay1007 Words   |  5 PagesThe Worst Hard Times by Timothy Egan conveys the story of farmers who decided to prosper on the plains during the 1800s, in places such as Texas, Colorado, New Mexico, and Oklahoma. They decided to make living, and some stayed during the worst droughts in the United States in 1930s. High temperatures and dust storms destroyed the area, killing animals and humans. This competently book reveals the prosperity for many, later revealing the time of the skinny cows. 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Think about what you have read so far in The Worst Hard Time: The Untold Story of Those Who Survived the Great American Dust Bowl. The entire text has a central idea, so does each section and chapter, as well as ea ch paragraph. Not only does a nonfiction text have a central idea, so does each section, chapter and paragraph. Let’s look at some example paragraphs from your book, The Worst Hard Time: The Untold Story of Those Who Survived the Great American Dust Bowl, andRead MoreSemester A Unit 3 Lesson 31626 Words   |  7 Pagesreading and what is it about? Think about what you have read so far in The Worst Hard Time: The Untold Story of Those Who Survived the Great American Dust Bowl. The entire text has a central idea, so does each section and chapter, as well as each paragraph. Not only does a nonfiction text have a central idea, so does each section, chapter and paragraph. Let’s look at some example paragraphs from your book, The Worst Hard Time: The Untold Story of Those Who Survived the Great American Dust Bowl, andRead MoreQuestions On Processor Demand Criterion1265 Words   |  6 Pages1990 (Lui Sha et al., 2004). In general, the processor demand in an interval [t1, t2] is the amount of processing time g (t1, t2) requested by those jobs activated in [t1, t2] that must be completed in [t1, t2] (Lui Sha et al., 2004). Hence, the feasibility of a task set is guaranteed if and only if in any interval of time the total processor demand does not exceed the available time, that is, if and only if (Lui Sha et al., 2004) Aperiodic task scheduling: The higher schedulabilityRead MoreThe Types Of Stereotypes789 Words   |  4 Pagesis never included in these labels, however true they may be.Given all this hate and judgement, people are better off keeping their worst traits to be dealt with by themselves and the people they are closestto, rather than proclaiming it to the public. The general public commonly will not help out one with such a big problem as their worst trait. Instead it is likely that they will go along shaming them just like the rest of the people around. Close friends and family would be the bestRead MoreGenerations: Baby Boomers1292 Words   |  6 Pageswe have the omniscient Baby Boomers (1946-1964), impious Generation X (1965-1979), and the judged Millennials (1980-2000: the youngest generation). With them, a futile attempt to convince non-believers that the Millennials are not, and far from the worst generation to exist. Just like the upper classman, the older generations say Millennials are â€Å"The dumbest generation†; â€Å"the intellectual future of the United States looks dim.† (Drutman). Criticizing Millennials use of the internet as a way to regurgitate