The trail of Tears Essay Example | Topics and Well Written Essays - 500 words

The trail of Tears - Essay Example Over 15,000 of our members, led by Chief John Ross, have signed a petition in protest of this false treaty. Despite this, the United States Supreme Court dismissed our concerns and this year ratified the treaty. We have now been given two years to migrate voluntarily to the west, away from our own country, to unfamiliar territory beyond the Mississippi. Forcible removal has been threatened if we do not leave on our own. Citizens of the United States, we do not wish to leave our lands, nor should we be forced to do so. Such a move would not be in our best interests; indeed it would be fatal to us as a nation. Firstly, it is an area completely unknown to us, and in addition is already occupied by other Indian nations who would not take kindly to encroachers upon their territory. We would be forced into close proximity with neighbors with whom we do not share a language or customs. The territory is also inadequately supplied with wood and water, making it much harder for us to survive. We appeal to the sense of fairness in the citizens of the United States, because previous to the false treaty, all agreements upheld our sovereign rights. Those treaties explicitly acknowledged us to be a separate people, in a separate territory comprising our own country, and were supposed to be secured and protected by your government. In the 1832 Supreme Court case Worcester v. Georgia, the majority opinion written by Chief Justice John Marshall rendered a judgment firmly upholding our rights. It stated, in part, that Indian nations should be regarded as distinct political communities, with their own territorial boundaries in which exclusive authority is exercised, as guaranteed by the United States. Although the case specifically addressed the legal question of whether the State of Georgia could forcibly seize any person residing within our nation with our permission, it forthrightly addresses our territorial and sovereign rights.

Ethics concepts Essay Example | Topics and Well Written Essays - 2000 words

Ethics concepts - Essay Example "Modern fertility treatments became the focus of much media attention in 1993 after the widely publicised case in which a 59 year old woman was enabled to give birth to twins by means of in vitro fertilisation with donated eggs and her partner's sperm. Fertility treatments raise a wide range of ethical and social issues" (Koch, 1993, p.143). Such factors as the potential child's welfare and interests are critical reasons for refusing to provide a couple with fertility treatment. In spite of the fact that the issue of conception is foremost in the present discussion, I would like to exemplify the situation with the process of child adoption. Even though it differs from conception, because the child already exists, authorities have established a number of criteria for adoption: parents' welfare, their personal qualities, health status and other information should be taken into account. These criteria are partially determined by supply and demand: for instance, potential parents are forced to compete with each other, because the number of infertile families is larger that the number of orphans who are to be adopted. Similarly, in vitro fertilization, associated with conception, poses following question: (bluntly speaking) will the child benefit from being born to these parents or would it be better if he/she never existed The likelihood of the particular potential child being born to another couple simply does not exist, and conception therefore is dissimilar to adoption in this sense. Naturally, it is hard to determine when it would be more preferable if the potential child didn't exist; the fundamental worth of an individual's life cannot be either measured or quantified, least of all if this life hasn't been started yet. It is possible to say, however, that the level of parents' responsibility would be rather low for it to be more favorable not to be born. Society's unwillingness to take care of a child excepting the most traumatic circumstances of horrible parenting proves this (Koch, 1993). Using the example of the 59-year old woman who gave birth to twins, a most important obstruction is that the mother will probably die when they are still at the stage of childhood, i.e. not having brought them up. "No doubt, other things being equal, it is preferable to have a mother who survives well into one's own adulthood. But to put this forward as a sufficient reason for denying fertility treatment is tantamount to claiming that it is better never to have existed than for one's mother to have died when one is still quite young" (Brindsen, 1992,p.280). In addition, in the case of in vitro fertilization, the interests of society are masqueraded as the potential child's interests. The procedure of selecting couples for the fertilization itself looks like the other official procedures that involve difficulties in distributing resources. There are two major hazards in failing to differentiate between the interests of the certain potential child and those of the potential children who might be born if resources were used to help other prospective parents instead. The first risk is that medical specialists may wrongly withhold the fertilization of the certain couple even if refusal to help them is not likely to bring benefit to other couples. The second hazard is that society may fail to support the process of

Management strategic-------Carry out a strategic analysis of the UK Assignment

Management strategic-------Carry out a strategic analysis of the UK grocery market using appropriate strategic management models - Assignment Example All these analyses have been carried out from the point of view of Tesco. However, references to UK general grocery market have been made times and again when required. The report concludes with the key takeaways from this detailed strategic analysis. 2. Overview of UK Grocery Market Retailing is one of the main contributors to UK economy with over 12% share. This sector acts as a bridge between production and consumption and is also a great generator of employment. In addition to the organized sector, there are a large number of unorganized players who are self-employed. UK grocery market has witnessed changes across both horizontal and vertical dimensions in the past few years. Horizontally, food retailers have ventured into other retail segments such as consumer goods, clothes, services and so on. Vertically, there has been a shift of power in the supply chain from the manufacturers to large organized retailers. This had a huge impact on the dynamics of the industry (Institute of Retail Studies 2003). 3. Internal Analysis of Tesco Tesco is primarily a food retailer with more than 2500 stores across the globe, a majority of which are in UK. The company has lately ventured into other business segments such as financial services, insurance, electrical appliances, telecommunications and insurance (Data Monitor 2004). SWOT analysis can be effectively used to carry out an internal assessment of Tesco. 3.1. Strengths One of Tesco’s biggest strengths is its continuously increasing market share. As of July, 2011, its market share is above 30% (Institute of Grocery Distribution). Tesco has almost doubled its market share in the past 7-8 years speaking high volumes of its growth. This growth has been enabled because of the continuous geographical expansion and opening of new stores. In addition, Tesco has strategically focused on non-food segment of the business and now it contributes very significantly to its revenues. Tesco has realized the importance of web i n its marketing. It has one of the biggest online supermarkets in the world. Tesco has a great brand image among its customers. It is known for high quality goods, innovative measures and efficient processes. Tesco’s lead over other players in UK market is huge. This has been made possible due to economies of scale it has gained over the years. 3.2. Weaknesses Tesco’s success story has largely been possible due to the UK market. However, it is susceptible to risk in case of change in government regulations. Tesco has a largely untapped market in emerging economies such as China and India. In addition, Tesco has been very aggressive in its expansion strategy and acquisitions. This has meant taking a large amount of debt affecting its balance sheets. Such a capital structure would work till the going gets good but may backfire in tough times. 3.3. Opportunities Tesco has opportunities from two perspectives. One is the geographical expansion in emerging markets as discuss ed above. The second opportunity is to make efficient use of its scale and venture into new business segments. The margins in the food industry are declining and it is profitable to consider new avenues. Tesco has already started working on the same with new segments such as skincare (Data Monitor 2004). In this way, it would also be able to realize economies of scope. 3.4. Threats UK grocery market has witnessed a lot of price wars. This has especially occurred due to entry of

Ethnic Marketing in Indonesia Essay Example for Free

Ethnic Marketing in Indonesia Essay Indonesia’s national slogan  « Bhinneka ika tungat.  » that means unity and diversity, is a strong local motto that celebrates differences, specially the demographic one. This slogan was adopted at independence in 1945. This can indicate in a first approach a strongly united population, a country where people are allowed to practice and conserve their ethnic Identity. But this never was the general case in Indonesia. There is this insidious ordinary racism in the country that takes place in their everyday living. Because this sacred diversity is however limited to ethnic groups with claims to a territory: this includes the Javanese, the Sundanese and the Madurese. There are other local groups that are not as significant. For the other groups that can not have the same claims, it is another story especially when it comes to the Chinese Indonesian. Being migrants with powerful and strategic positions, they are often excluded by the population or they exclude themselves. If this is how the Indonesian society can be perceived, it isn’t the same when it comes to the marketing approach. It can be said that it is not that simple to talk about ethnic marketing in Indonesia when it is related to physical characteristics. If the comparison was made with Singapore, It is much easier to identify that a person belongs to a certain ethnic group when she is shown in an advertisement. It becomes more delicate when the ethnic groups have a lot of similarities. Even if Chinese, Indo or Arab origins Indonesians are not accepted everywhere or not recognized as true Indonesian by a part of the society, they still have their place and are represented for marketing purposes. We can give an example of Western Union: For the first Ad, adressed to the chinese Indonesian poulation, the person is an office working man, we would rather think that he has a good salary and is educated. The emphasis here is about the importance of the speed of the transaction. For the second Ad, more adressed to Indonesians with claims of territory, The main character is a little girl that due to the received money, could go to school. It shows the importance that is taking girls education in those groups. When it comes to the language, companies that operate at a national level generally use Indonesian or English for their advertisement campaigns. The use of a specific language related to a group is more likely to stay in the frame of small companies that operates in very restricted areas.

Sample Reflection Assignments Essay Example for Free

Sample Reflection Assignments Essay Written assignments are of two types: Reflections and Project Reports. Reflections: An essential element for student learning in service-learning courses is written and oral reflections on the field study experience, as well as on other elements of the course. To have an experience isn’t enough to ensure learning; you need to intentionally and thoughtfully reflect upon the experience and what you learned from it to ensure that learning occurs. As T.S. Eliot once wrote (The Four Quartets, New York: Harcourt Brace, 1943, p. 24), you can â€Å"have the experience but miss the meaning.† Towards this end there are eight written reflections required in this course. These reflections are intended to help you focus in on what you are learning (or not learning), identify issues or concerns on your mind, explore an idea more deeply, or in other ways enhance your learning about the course topics, yourself, and others. According to the North Carolina State Service-Learning Program (Reflection and Articulating Learning, Faculty Center for Teaching and Learning. NC State, 2004) â€Å"the best reflection follows a simple three step model: (1) describe the experience(s) objectively, (2) analyze the experience(s) in terms of the categories of service-learning objectives (personal, civic, academic), and (3) articulate (express) the learning that results. We will go over these steps as part of our first reflection exercise. These reflections should be approximately two pages long (500 words). They will be graded. The best six out of eight grades will be used towards your final grade. Evaluation of reflections will use a set of criteria (rubric) to be distributed and discussed in class. Project reports: There will be a series of written assignments related to your service-learning project. In preparation for the write-up of the final project report for the community partner (along with other possible final products like videos, curricula, maps, etc.), students will prepare a written work plan and two progress reports (schedule to be provided). All of these written assignments will be graded and become part of the final grade. Details on all of these written assignments will be provided and discussed in class. COURSE: Cultural Health Care (NURS 105) INSTRUCTOR: Rycki Maltby PhD, RN, FRCNA Final Paper (25%) The purpose of this paper is to synthesize your learning over the semester. The paper should be 8-10 typed (word processed) pages, 12 cpi, and follow the attached guidelines for written assignments. Using the service-learning project, the cultural self-study, and the in-class reflections, discuss what you have learned. What surprised you? Has your thinking/perspective about particular issues changed? Did you become aware of the subjective nature of value judgments? Why or why not? Can you find any support in the literature for your position? Provide evidence of at least four articles. What is the next step for you in learning about cultural health care? Be sure to provide examples from your practice and tie in literature. Guidelines for Written Assignments ‘A’ Level 1. Introduction is comprehensive, gives reader good direction, ‘sets the scene’, and is followed throughout paper. 2. Summary/conclusion is thoughtful and relevant. 3. Fundamental issues addressed in depth with original arguments and critical judgements, demonstrating insight and creativity. 4. Current and classic primary literature sources are utilized. 5. Writing style shows evidence of individuality, unity and fluency. 6. Overall presentation of the paper is  professional with no errors in syntax, spelling, etc. (i.e. Proper English language usage), and follows APA format. ‘B’ Level 1. Introduction gives reader direction and is addressed throughout the paper. 2. Summary/conclusion is clear and concise. 3. Fundamental issues addressed with evidence of some original arguments and critical judgments. 4. Current literature sources utilized (primary and secondary) 5. Writing style is fluent with evidence of individuality and clarity. 6. Overall presentation of paper is neat and well organized with few minor errors in syntax, spelling, etc. (i.e. Proper English language usage), and follows APA format. ‘C’ Level 1. Introduction gives reader direction. 2. Summary/conclusion is clear and concise. Fundamental issues described but limited originality of arguments and few critical judgments. 3. Limited literature sources are utilized (current and/or classic). 4. Writing style is fluent and some evidence of individuality and clarity. 5. Overall presentation of paper is neat and minor errors in syntax, spelling, etc. (i.e. proper English language usage), and follows APA format. COURSE: Adv. Development Sem.: Communities and Human Development (PSY 366) FACULTY: Lynne Bond Final Writing Assignment: At the end of the semester, you will be assigned a final writing assignment. The purpose of that assignment will be to help you integrate the reading, writing, discussion, and data gathering, analysis and interpretation we have done during the semester. The exact format of that assignment will be shaped, in part, by the development of our community-based project. As we progress throughout the semester, we will work together to develop more explicit guidelines for this project. COURSE: Community Psychology (PSY 295) FACULTY: Lynne Bond Analytical/Critical Reflection (2 pages): These brief papers are to focus specifically upon the week’s reading assignment and its relation to our community field work, other readings, and personal experience and observations of your own. Your goal is not to summarize the readings. Rather, you should analyze and reflect critically upon a couple significant issues raised in the readings. To reiterate, ideally you will critically reflect upon these readings by linking them with other material we have covered in our course (readings/discussion), community field work you have done, and other aspects of your experience and knowledge (e.g. personal history, issues observed in media, other courses†¦etc.). We will discuss and practice â€Å"reflection† activities in class at the beginning of the semester to help you feel more comfortable and prepared to complete these reflection papers. Intro to the Nonprofit Field (Carrie Williams Howe) Final Reflection Assignment: WHAT? What have you learned through this course? What stands out to you, feels new to you, excites you, or challenges you? SO WHAT? Why does this learning matter? Why does it stand out to you? Why is it important to you personally or in the bigger picture? NOW WHAT? What do you do from here? How will you take this learning with you? Does this learning change your perspective, your career goals, or your interests? Your reflection should be 3-5 pages long and should dig deeply into this experience to reflect on your learning. It should connect to academic concepts and critically examine your own development through this learning. You will be graded based on the reflection rubric provided in your syllabus. During Service Reflection: Critical Analysis Papers (9): You will be asked to write a brief critical analysis paper for each topic we cover in this course. These papers should thoughtfully connect your experience at your nonprofit to what you are learning from readings and course notes. These reflections should be about 2 pages and will be graded based your ability to integrate the following: Information about your Nonprofit – the actual information about your nonprofit in relation to the topic we are studying (list of board members, for example), and an examination your own experience in learning about or being involved with this element of the agency. Your synthesis, interpretation, and analysis of the above factors with course information (for example – does it align with the reading? Does it seem effective? Might you suggest changes or improvements?).

Human Computer Interaction Assignment English Language Essay

Human Computer Interaction Assignment English Language Essay Technology has had a big impact on society, as it has made many drastic improvements to the way life in general has changed and improved so much. For instance, technology has improved usability of software and hardware in society. In addition specialised interfaces have helped people with disabilities enabling diversity to take place. Describe the impact of HCI on society, the economy and culture, providing 5 (five) examples of each topic. Society Touch screen phones, using phones is now easier because of the touch screens Input/output Game controllers, game controls allow you to play a game and connect with others by sending messages or attaching a microphone to it to talk Remote control, allows you to remotely control something without being right next to the thing you want to control Head up Display, would be good for drivers since they wont have to look away from the road to see how fast theyre going Economy Text readers, also known as text to speech Voice input, example telling a lamp to switch off and it switches off Thought input, wheel chair users will have the ability to control their wheel chair by just thinking about it Mobile communication, ability to call or text anyone world wide Reduced complexity of input, everything is simple and requires no more than 2-3 options of input Culture Games, allow people to connect with each other allowing one another to have an open mind and talk to different people with different opinions and thoughts Deskilling work, androids/robots will replace the easy things humans do, e.g. at tesco and a few other places you can serve yourself and self-checkout Mobile entertainment, youre now able to watch videos about different cultures with your mobile Laptops, ability to search the internet, interact with others, watch videos Domestic appliances, things like microwaves diswashers Task 2 (P2) Considering that the HCI design principles create a big impact on a users experience of a hardware and software product, explain the Schneiderman (eight rules) and Nielsens ten usability heuristics principles. Remember to explain perception, behaviour models and information processing. Schneiderman Strive for consistency: either it be the consistent same lay out or colours, or the identical terminology used in menus, prompts and help screens, if it is in a similar situation it should all be the same or somewhat similar. Enable frequent users to use shortcuts: A regular user of a device or a certain type of application should have the ability to have a shortcut to things they use the most; this would speed up the work flow. Offer informative feedback: When a user is interacting with their device or application, they should receive feedback that gives them a clear representation and is also understandable. Design dialog to yield closure: A users gets a good feeling when they know there is a flow to what ever theyre doing, that everything is in a chain, one thing has to happen in order for another. For example, sending a message on an application such as whatsapp first you need to add the contact, then the message its self, and then after youve sent the message it shows a D letting you know its been delivered. Offer simple error handling: If something goes wrong with a device, a simple type of hint should pop up to help the user with the problem that occurred. If a certain app crashes it should ask the user if theyd like a error report to be sent. Permit easy reversal of actions: In other words undo this is needed in a HCI because it would prevent any permanent mistakes. Support internal locus of control: a user should receive some what a type of display letting the user know that something is actually happening. Lets say a user attempts to open a app and the app is taking a few seconds to get ready and open up, the user would instinctually think the app didnt respond to his/her touch so he/she will attempt to reopen the app multiple times until the app malfunctions. Reduce short-term memory load: When a user is using a device they shouldnt be thinking about the amount of times theyll need to push a button or what not, it should be a simple click of a button, and with menus it shouldnt require a button push to go up or down it should be a simple scroll bar. Nielsens ten usability heuristics principles Visibility of System Status: The system should have a certain type of way to let the user know what is happening and with a good time. Match between system and the real world: The system should be able to speak the users language, with the correct words and the right concepts and with the right time, so it is communicating clearly with the user User control and freedom: Sometimes when youre using a device in a rush you tend to open random applications and then you have the ability to instantly abort the application, without having to go through the process of the extended dialogue. Consistency and standards: The system should always have continuity across the platform. Error prevention: The better the design is the one with the better error recovery, but also prevents users from making the same errors twice. Recognition rather than recall: The user shouldnt have to remember the information from dialogue to another, it should all be recognisable and visible to the user. Flexibility and efficiency of use: The system should have accelerators, which are unseen to the regular user but they allow the expert user to do things and navigate faster with more frequent actions. Aesthetic and minimalist design: Dialogues shouldnt contain information that is not needed in that specific area, you should always make sure your system is efficiently composed. Help users recognise, diagnose and recover from errors: Error messages and error prompt boxes should explain the problem in plain language without code, and should also precisely indicate whatever the problem may be and also give advice to the user on a solution. Help and documentation: Although a system is better without documentation, it might be necessary to provide the help and documentation, so that any such information should be easily searched for and found. It should also be focused on what the users task is and list the steps to be carried out. Task 3 (P3) McCann Phones a small and up and coming new mobile phone producer has decided to develop a new mobile phone interface for its next series of mobile phones to replace its highly successful first range. The developers have come up with three possible interfaces and they need to decide which one to use. It has been decided to carry out an investigation on the alternatives prior to production. You have the support of the software engineers and production manager so prototypes will be made available to you. As you are the resident usability evaluator it is your task to carry out any necessary survey work and to come up with proposal. Here are the three possible interfaces: The convention qwerty keyboard as used in most computer systems. An alphabetical keyboard (a, b, c, d, e,,z) A keyboard based on frequency of character us (e, t, a, o, n, rz) Design the input and output of your mobile prototype. Choose one of the suggested interfaces and explain why. F:Unit 23 HCITemplate of phone design.jpg Clicking either one of these buttons will open up a certain app/webpage, depending on the one you click, for instance if you click on the one with f will open up a Facebook app or webpage. Reason why Ive added this to my mobile prototype is because people now use these two to communicate with each other, with the button input being there, itll allow you to get in contact faster and easier considering all youd have to do is click a button. Task 4 (P4) The knob in the centre of this washing machine system controls the different fabric washing systems and the settings for rinse and spin. The control does not have a stop in either direction; in other words it continues to go all the way round to the next cycle. Create input and output for this machine which would be best for adjusting the cycle according to the fabric, temperature, speed and timer. You could draw a picture to help you explain the reasons. Provide two new ideas that are not available in the industry. img_0193.jpg Below is what my design would look like and I will be explaining the two things that Ive implemented into my design that havent been used in the industry. 2. Touch screen, everything you input will be touch done via the new sleek touch screen design. Child lock passcode, this will be programmed in a way that it will be child proof, for example if you want to lock your machine after it has started and keep every button locked, youll enter the child lock. It is designed in a way so that no matter what you push nothing will be happening but to deactivate the lock youll first have to enter the first 2 digits right, this will then enable you to see what youre inputting and then you enter the rest of your passlock to unlock the machine. G:Unit 23 HCITask 4 Design 1.png Task 5 (P5) Based on the case scenario before, explain how you would test your system according to its functionality. Comment on the usefulness of such system and point out any drawbacks. Testing the system child lock To test the system youll first have to enter a child lock pass lock and then attempt to push any of the touch screen buttons and see if anything happens, and nothing should happen if the system is correctly made. This will be useful considering little children love to stand beside the washing machine when its spinning and tend to push buttons, but with the child lock they wont be able to push any buttons. The downside to this system is that the password is 4 digits long and to have half access to the system youll have to correctly enter the first 2 digits otherwise youd just be pushing the keys and nothing would be happening, so the drawback would be if you forgot the passcode you would have to end up calling the company to come and reset your password. Testing the touch screen To test this youll have to push what you want, for example, cotton, with 50 degrees Celsius and then on high spin, if the system is working properly it would input the material first and then the temperature and then lastly the amount of spins it will be doing. The touch screen with the options is useful because its simple to use and you can adjust it to the way you want it and wont have to clock the washing machine knob to the way you want it, you just have to input the things you want with a touch of a button. The big drawback to having a touch screen is that if it is dented or broken it will be not be operating properly, it probably wont even be operating at all, so you wont be able to input anything. Task 6 (P6) Bionic vision was once the preserve of futuristic technology shows. However, those Tomorrows World days could soon be reality with a pair of glasses linked to a computer offering hope to thousands of visually impaired people in Britain. Current technology that can give profoundly visually impaired people a form of sight through a retinal implant is expensive and invasive, so developing an affordable, non-invasive alternative would be a welcome improvement. And this is exactly what scientists at the University of Oxford are working on. The technology works by having a tiny video camera mounted on to a pair of glasses, which relays information to a small computer in the users pocket. The computer recognises objects or people and relays the information to the lenses via tiny LED lights. (Light-emitting diode). People would see a bright light within the lenses themselves to indicate an object. The brightness of the light would then indicate how close, or how far away the object was and that may be enough for the wearer to navigate their way around. Using a computer to interpret the world on behalf of the wearer means we could use different coloured lights in the lenses to allow different types of information to be fed back to the wearer explains Dr. Stephen Hicks, research associate in the Department of Clinical Neurosciences at the University of Oxford. The advances in mobile phone and computer game technology, such as face recognition, tracking software and depth sensors enable the visually impaired to access a whole new host of possibilities. (Extract from Metro, September 19, 2001) Explain how you would design the device and document how you would consider the input, output, the quantitative measures and qualitative measures of effectiveness of your product. This would be designed on a pair of glasses with a lens that is 5cm thick, it would need to be sort of thick to fit in a pair of small LCD screen into each of the lens. The input and out put would work in a way with different bright colours, for example if the area is clear of objects and people it would be a black light with a blue light highlighting the walls so that the user of the glasses doesnt bump into the walls, and if the user is near people it would be a black light with green light highlighting approximately where the object or person is so that the user doesnt bump into them. If its dark at night instead of the backgrounds being black they would be white and objects, people and walls would be highlighted with purple. The glasses would also be implemented with a vibrator and a sound card to make beeping noises when someone gets close to an object within a close radius of 50 centre meters, and depending on how low/high the object is, it will make a beep vibrate, the user has the ability to switch one of these of. Each beep and vibrate will have a different meaning for example if a object is really close to the person and they can walk over it, it would simple beep twice and vibrate twice, just in case they didnt hear the beep. C:Usershijh0736Pictures48416_1 copy.jpg Ear piece attached to the glasses that goes into the users ear, it sends beeping signals depending on the diameter of how close they are to a object, they can get the settings changed from where they where given the product, just in case the beeping gets annoying or they want to change the frequency of the beeping. LCD screen projecting lights, black background, with green to represent a object. If the person that is vision impaired isnt impaired badly, then they can ask someone to change the settings of the screen so that the black background is taken away and it is just see through but it would still highlight objects as green. If the person that is vision impaired is badly impaired, no need to worry their is a ear piece that also helps, with beeping when coming in close contact to a object. Submission Date: Week commencing, 22nd October 2012 in class Failure to submit on time is a serious concern. Please read your Rules Regulations. Any request for EXTENSION needs to be made in writing to the subject lecturer and copy to Tutor. GUIDE LINES FOR COMPLETING THE ASSIGNMENT Please attach the complete cover sheet with your coursework. Make sure your read all of the tasks in this assignment before you start. Create your assignment in one Word document. Please write in your own words. Proof read and spell check your work. Reference your work by providing bibliography/source table on a separate sheet in the same document of all the books, websites, notes, etc. Ensure the assignment presentation is clear and that the tasks are identified with the appropriate task numbers and the grading criterion. Include your name in the header, unit no, title, appropriate file name and path, page numbers in the footer. Submit on time via Moodle (final submission). Save a copy of your work on the WKC server this is important and a requirement. Presentation of your Assignment The work will use correct paragraphing, formal grammar, tenses and spelling. The pages of the assignment will be consecutively numbered. The correct cover sheet must be submitted with your assignment. Assessment Policy BTEC Courses Students will be permitted to go through a draft with the lecturer during a workshop determined by the lecturer. If you do not attend this workshop, your draft will not be checked. They will then be expected to hand in the assignment on the due date and time as specified on the assessment brief and the AR1 (assessment plan). Late submissions are capped at a Pass unless you have extenuating circumstances and have explained these to the tutor prior to the submission date. You are entitled to one resubmission with the Pass criteria in the closed book assessment For the Merit and Distinction criteria, anybody who is unable to achieve on first submission will be require to sit a closed book assessment at the end of the semester. REFERENCING and BIBLIOGRAPHY The Harvard Referencing System will be used conventionally to identify all secondary research A conventional and complete Bibliography using the Harvard Referencing System will be included. Please note that you are NOT allowed to include any reference from the Wikipedia website your assessment may be adversely graded if Wikipedia features in the referencing or Bibliography. Please see the example below for how a Bibliography should be formatted: Example Bibliography Books Grbich, C. (2007). Qualitative Data Analysis: an introduction. London, Sage Publications Ltd. Hart, C. (2005). Doing your Masters Dissertation. London, Sage Publications Ltd. Journal Articles Abdullah, F. (2006). Measuring Service Quality in higher education: HEdPERF versus SERVPERF. Marketing Intelligence and Planning 24(1): 31-47. Bignold, D. (2006). Schooled and Tooled. Caterer Hotelkeeper 10th August 2006: 23-26.

Single Chain Antibody Production

Single Chain Antibody Production ABSTRACT The study aimed to characterise His- and Myc- tagged scFv MFE-23 antibodies produced from transformed E.coli cultures using ELISA and immunohistochemistry assays. Anti-His, anti-myc and anti-MFE secondary antibodies were used in the CEA/PBS coated ELISA plate with horseradish peroxidase-OPD chromatic reaction for detection. Culture 1 was identified to produce MFE-His and culture 2 giving MFE-Myc antibodies. The immunohistochemistry assay confirmed the CEA binding profile of scFv MFE-Myc by the comparison between negative controls, positive anti-CEA binding reactions and 4-stage anti-Myc binding of the scFv MFE-Myc examined. The CEA specificity displayed by tagged scFv MFE-Myc can be utilised in antibody-based cancer therapy by targeting tumour antigens specifically. INTRODUCTION Cancer arises due to the defective regulation of normal cell proliferation and homeostasis. This allows tumours to possess the capabilities including self-sufficiency in growth signal, insensitivity to antigrowth signals, avoidance of apoptosis, unlimited replicative potential, sustained angiogenesis and metastasis.1 In additional to the surgical removal of tumours, conventional cancer therapies such as radiation, chemotherapy and immunotherapy have a focus in inducing cytotoxicity against malignant cells. With better understanding of the molecular biology of carcinogenesis, targeted therapies are being developed to achieve lower toxicity to normal tissues and higher clinical efficacy through disrupting pathways that contribute to the tumours proliferative advantage. Attempts are also being made in developing cancer gene therapies to compensate or repair the mutated genes. All tumours express their unique set of antigens on cell surface which can be a result of genetic alterations, upregulated self-antigens or tissue-specific antigens that can be utilized to distinguish cancer cells from normal cells. Antibodies, one of the effectors in immune response, are Y-shaped proteins that each recognizes and binds to specific antigen (Figure 1). The protein consists of two light chains and two heavy chains, with variable (Fab) and constant (Fc) domains on each of the chain. The region of the antibody at which antigen binds is referred as the Complementary Determining Regions (CDR) present at the variable Fab region. The Fc region is responsible in modulating immune response through activation of the complement cascade and Fc receptor mediated activation of effectors such as phagocytes, mast cells, neutrophils and Natural Killer (NK) cells.3 The initial approach of antibody-based cancer therapy is to tag cancerous cells as foreign and eliminate such targets thro ugh cytotoxic effectors of the human immune system. Antibodies against essential growth factors can be synthesized to sequester such molecules from further promoting tumour growth. The high specificity of antibodies to particular antigens also serves as a vehicle in delivering killing machineries to tumour cells. For example, antibodies can be directly conjugated to radionuclides, toxins or cytokines, or indirectly to the surface of liposomes carrying drugs or toxins.4 Antibody-directed enzyme prodrug therapy (ADEPT) is also available in which the antibody targets an enzyme selectively to the tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug.4 These various strategies aim to minimize the systemic toxicity afflicted by the cytotoxic agents administered. Monoclonal antibodies are usually preferred compared to polyclonal ones, as they recognize specific epitope of an antigen and hence have greater specificity. Carcinoembryonic antigen (CEA) is first identified as a glycoprotein in the human colon cancer tissue extract and fetal gut, and plays a role in cell adhesion.5 Although CEA can also be detected in normal gastrointestinal tissue, the glycoprotein is overexpressed on the plasma membrane of colon cancer tissues.5 CEA level has also been found to be highly elevated in various cancers of an epithelial origin such as breast, lungs and pancreas.5,6 Moreover, normal CEA is localised on the luminal surface of columnar epithelial cells lining the crypts of the intestine so the glycoproteins are not directly accessible to the blood flow.6 However CEA is usually found on all sides of the cell membranes in tumours. Thus, CEA can be a useful target on cancer cells in immunotherapy using anti-CEA antibodies. The objectives of this lab practical are to introduce the applications of antibodies in cancer therapy and diagnosis through immunohistochemical staining of tissues. It also serves to familiarise students with the enzyme linked immunosorbent assay (ELISA). This is achieved by the production of MFE-23 single chain Fc antibody fragment (scFv) against CEA from transformed E.coli cultures. The scFv MFE-23 can be linked to either a His- or a Myc- tag. The unknown antibodies are characterised in the ELISA assay using appropriate detection antibodies, and the chromatic reaction between horseradish peroxidise (HRP) and OPD substrate. The scFv MFE-23 obtained by students is also used in the immunohistochemical characterisation in cryostat sections of normal and cancerous human tissues. MATERIALS METHODS E. Coli growth curves E.coli cultures were transformed with pUC119 containing either His- or Myc- tagged scFv MFE-23 (Culture 1 2) and were incubated overnight. The expression of pUC119 was controlled by the lac operon, which could be induced by either lactose or lactose analogue isopropyl-1-szlig;-D-thiogalactoside (IPTG). The vector also encoded for ampicillin resistance. The E.coli cultures were grown with ampicillin selection and 0.05% glucose. Optical density (OD) readings at 600nm were taken at 30min interval until OD=0.9 when IPTG was added to both cultures to induce pUC119 expression and hence scFv MFE-23 production. A negative control of 2xYT was set up, and all three cultures were incubated at 30? overnight. Full experimental procedures are described in Appendix 1. ELISA assay The ELISA assay aims to characterize the identities of the tags conjugated to scFv MFE-23 obtained in the supernatant of the overnight E.coli cultures. Detailed protocols of the assay can be found in Appendix 1. 36 wells of the 96-well ELISA plate were coated with the CEA antigen or phosphate-buffered saline (PBS) as its negative control. Supernatant of the overnight E.coli cultures 12 from the bacterial growth curve assay was obtained which contain either His- or Myc- tagged scFv MFE-23. The supernatant samples 12, the positive control MFE-his-myc antibodies and the negative control 2xYT growth medium were added to the corresponding wells as indicated in the format diagram in Appendix 2. Secondary antibodies rabbit anti-MFE23 polysera, mouse monoclonal anti-HIS tag (TetraHis, Qiagen) and mouse monoclonal anti-MYC (Sigma) were added to the corresponding wells (Appendix 2) to bind the primary antibodies present. Tertiary horseradish peroxidase (HRP) conjugated antibodies against the secondary antibodies goat anti-rabbit HRP (sigma) and sheep anti-mouse HRP (Sigma) in blocking solution were added to the wells (Appendix 2). OPD substrate buffer was applied to each well to detect the presence of HRP, which should give a yellow-orange product in case of positive result. HCl was added to stop the reaction when colour has developed, and the OD at 490nm for each well was measured. Immunohistochemistry assay Five glass slides, each containing 2 colonic adenocarcinoma, 1 normal colon and 1 normal liver tissue sections were fixed and processed for immunohistochemical staining. Avidin-biotin-peroxidase complex (ABC complex) was added following the application of biotin-labelled antibodies. The localisation of antigens was visualised by the formation of brown pigments, as peroxidise reacts with the diaminobenzidene (DAB) substrate. Lattices of several peroxidase molecules were formed to amplify the binding signal from the biotinylated antibody.7 A summary of the treatment given to each slide was illustrated in Table 1, and the complete protocol can be found in Appendix 1. RESULTS E. Coli growth curves Both E.coli cultures transformed with either His-tagged scFv MFE-23 or the Myc-tagged version in a pUC119 vector follow a similar exponential growth curve, as shown by the plot of OD600 against time (Figure 2). The bacterial cultures were in lag phase at t=0-90, and the log phase from t=90. It took approximately 175minutes for the cultures to reach OD600=0.9. The PBS negative controls in wells A-F/7-12 worked relatively accurately with low OD490 readings. Most readings also corresponded to the negative controls in the CEA coated wells (B4-6, D4-6, F4-6) but with 2xYT added instead of primary antibodies. However, the OD490 readings for wells C10-12 and E10-12 were higher than most readings from the negative controls. The higher OD490 readings obtained in certain wells in comparison of their corresponding negative controls indicated the presence of yellow-orange product formation from reaction between HRP and OPD substrate. These positive outcomes found in certain wells are highlighted in the shaded cells of Table Immunohistochemistry Assay Figure 4 shows the immunohistochemical binding reactions for the negative controls (slides 3-5). Slide 3 acts as a negative control for slide 1, treated with the 4-stage anti-Myc technique but omitting the primary Myc-tagged scFv MFE-23 antibody. Thus we should not be able to visualize the localization of CEA antigens due to the absence of MFE-23 binding on slide 3. Slide 4 is a negative control for slide 2, omitting the mouse monoclonal anti-CEA A5B7 antibody treatment in the 3-stage mouse monoclonal technique. No binding reaction is expected on slide 4 as well. Slide 5 was treated like slide 4 but without initial biotin/avidin blocking. As expected, the colonic adenocarcinoma tissue section of slide 3 does not display brown colouration and hence there is no binding reaction to the cytoplasm of tumour cells and connective tissues. Binding reaction to the cytoplasm of cryptal epithelium is not seen for the normal colonic mucosa, although there are strong brown colourations for a few cells in the lamina propria. The normal liver tissue on slide 3 shows some weak reaction with the parenchymal cells. Similarly, the parenchymal cells of the normal liver tissue on slide 4 do not display brown colouration and hence indicates the absence of binding reaction. On slide 5, the normal parenchymal cells are positive for binding reaction, demonstrating the presence of biotin in normal liver. Figure 5 shows the immunohistochemical binding results of slides 1 and 2. Slide 1 was treated with the 4-stage anti-Myc technique and slide 2 is the positive control treated with the 3-stage mouse monoclonal technique (See Table 1). For the colonic adenocarcinoma tissue on slide 1, strong brown colouration is present in the cytoplasm of tumour cells and the basement membrane of malignant acinar structures. Weak positive binding reactions can also be observed in fibrovascular stroma. The normal colonic mucosa of slide 1 shows strong reactions to the cytoplasm of goblet cells in the cryptal epithelium as well as a few cells in the lamina propria. The normal liver tissue shows only weak positive reactions with the parenchymal cells. Thus the scFv MFE-myc antibody was reactive with both normal colonic epithelium and adenocarcinoma, but not the biotin/avidin blocked liver tissue. The colonic adenocarcinoma tissue on slide 2 shows strong reactions with the tumour cell cytoplasm and the basement membrane of malignant acinar structures similar to the reactions seen in slide 1. Weak positive results are obtained in fibrovascular stroma of the positive control slide. As the binding reaction of slide 1 was similar to that of the positive control, this confirms the CEA reactive profile of the scFv MFE-myc antibody from the E.coli supernatant sample 2. DISCUSSION E.coli Growth Curve Both E.coli cultures followed the exponential growth curve as expected. However, the growth curve was obtained in the absence of a negative control i.e. same volume of 2xYT to be treated in the same way as the two cultures. The lack of a proper negative control means that the possibility of contamination cannot be eliminated. Thus it is unknown whether the increase in OD600 readings was partially attributed to culture contamination. ELISA Assay As mentioned in the results, the OD490 readings for wells C10-12 and E10-12 were higher than most readings from the negative controls. This might indicate contamination of these wells with CEA antigens, or insufficient PBS washing following the application of HRP-conjugated antibodies. Wells A-B/1-6 were applied with anti-His secondary antibodies and so would indicate the presence of His-tag by the production of yellow-orange product. Wells A1-3 were treated with supernatant from bacterial culture 1 and wells A4-6 with that of culture 2. As higher readings of OD490 in the positive control B1-3 and wells A1-3 were obtained compared to the negative control B4-6, the culture 1 supernatant contained the His-tagged scFv MFE-23. Wells C-D/1-6 were applied with anti-Myc secondary antibodies and so would indicate the presence of Myc-tag by the production of yellow-orange product. Wells C1-3 were treated with supernatant from bacterial culture 1 and wells C4-6 with that of culture 2. As higher readings of OD490 in the positive control D1-3 and wells C4-6 were obtained compared to the negative control D4-6, the culture 2 supernatant contained the Myc-tagged scFv MFE-23. The anti-MFE antibodies added to wells E-F/1-12 can bind to both His- or Myc-tagged scFv MFE-23. Thus binding should occur against primary antibodies from both cultures 12 and also the positive MFE-myc-his control, as demonstrated by the higher OD490 readings in wells E1-6 and F1-3 compared to the negative controls F4-6. It can be observed that the anti-His antibody gave a stronger signal than anti-Myc and was due to anti-His binding more strongly to its target than anti-Myc (Unpublished results, Kogelberg, H.). Comparing the OD490 of wells E1-3 and E4-6, absorbance of anti-Myc was slightly higher than anti-His despite the lower binding affinity of anti-Myc. Thus there might be a higher concentration of MFE-Myc in the culture 2 supernatant than MFE-His in culture 1, although technical issues like washing times, salt concentration and pH, or structural characteristics affecting the accessibility of antibody can affect the amount of binding. Immunohistochemistry Assay The results obtained from slides 1 and 2 confirm the CEA reactive profile of scFv MFE-myc antibody, as both slides displayed similar binding reaction patterns in colonic adenocarcinoma. The weak positive signal in normal liver tissue in slide 1 is likely to be caused by cross-reaction of secondary mouse anti-Myc antibodies, as similar result can be observed in the negative control (slide 3). Primary antibodies may also cross react and bind to non-target tissues. This demonstrates the possible cross-reactions with antibodies and hence the importance of negative controls to eliminate such artifactual reactivity. The strong binding to goblet cells cytoplasm in cryptal epithelium of normal colonic mucosa in slide 1 is consistent with the findings of CEA present in normal colonic mucosa.5 Although CEA level is lower on normal colonic mucosa, their presence implies that antibodies against CEA in cancer therapy may target normal cells other than malignant cells. Thus it is important to control anti-CEA antibody concentration used to avoid imposing toxicity to normal tissues yet is effective in producing a clinical response on cancer. The negative controls (slides 3-5) allowed the assessment of the level of binding for secondary, tertiary and quaternary antibodies/reagents. The slides also revealed any endogenous background material that might be confused with specific binding of primary antibodies, as well as information about the basic pathology of tissues. The inclusion of normal liver sections helped illustrate the importance in carefully controlling the specific reactions of test antibodies with potential targeting specificity for other cellular proteins. The ABC complex for detecting biotinylated horse anti-mouse antibodies can also react with biotin present in liver and give a positive non-specific result. This unwanted reaction as seen in slide 5 can be prevented by biotin blocking treatment of tissue samples. Moreover, myeloperoxidase white cells containing endogenous peroxidase can react with the DAB substrate and produce brown colouration even in the absence of ABC complexes. The normal colonic mucosa in slide 3 provided examples of such artifact. The naturally occurring bile pigments in liver seen as green/brown granules under high-power bright field microscopy can also be found occasionally. Careful interpretation of slides is required to avoid false judgments as there can be false positive reactions. In addition, it should be noted that adequate fixation of the sample is important in successful localisation of antigens present and hence an accurate representation of antigen distribution profile. The procedure helps to ensure the preservation of tissue morphology, the immobilisation of antigen and the preservation of antigen immunoreactivity. It is also important to ensure optimal fixation for an adequate permeability of the tissue to the immunochemical reagents. Other than mmunohistochemistry, western blotting and immunoprecipitation (IP) can be carried out to confirm scFv specificity. Antigens transferred to nitrocellulose membrane can be probed by specific antibodies in western blot, or precipitated out of lysate using antibodies in IP. Affinity chromatography can also be used which is a method of separating biochemical mixtures based on highly specific biological interaction. Specific antigen can be covalently coupled to a solid support and allow supernatant with the testing antibody to flow through so that only specific antibodies will be bound to the antigens. The assay used in this study can only detect antigens in a non-quantitative way with chromatic display of antigen localisation. Instead radioimmunoluminography (RILG) can be used for quantitative measurements of antigen concentration along with its distribution in histological sections.8 Radiolabelled antibodies against specific antigen can be applied to tissue sections and bound antibodies are mapped by phosphor imaging. Radioactivity detected in each pixel of the digital image will be proportional to antigen concentration if saturating antibody concentration is used. Future Perspective of Antibody Targeted Cancer Therapy The successful application of scFv MFE-23 with a Myc-tag at its C-terminus for detection in immunohistochemistry proves that the attachment of small molecules to the antibody will not affect its specificity for CEA. Thus the scFv chain can be conjugated to cytotoxic reagents or to be used for ADEPT as mentioned previously. The potential clinical efficacy of scFv MFE-23:enzyme fusion protein has been shown in nude mice with human colon adenocarcinoma xenografts by the Bhatia group.9 Moreover, scFv MFE-23 can be used in radioimmunoguided surgery (RIGS) based on the pre-operative injection of a radiolabelled anti-tumor antibody to detect tumour deposits during surgery. A Phase I clinical trial of RIGS using 125iodine-labelled MFE- 23-his scFv has reported good selective localisation at sites of primary colorectal cancer and metastases.10 As illustrated by MFE-23 scFv fragment, antibody targeting has the potential for selective imaging or delivery of anti-cancer molecules. Antibodies can be engineered to modify their biological properties with increased specificity and functionality. This is achieved by reducing antibody size, altering valency, and fusing to different molecules to improve therapeutic efficiency. Scientists have been trying to produce smaller antibody fragments but retaining specific binding to antigens, in order to minimize immunogenicity and achieve better tumour penetration. Continuous research on the specificity and stability of these fragments, and hunting for more tumour-specific antigens are required to further expand the field of antibody-targeted cancer therapies. Slide 3 is a negative control for slide 1 which was treated with the 4 stage anti-Myc technique. Slide 4 is a negative control for slide 2 which was treated with the 3 stage mouse monoclonal technique. Slide 5 is a negative control for slide 2 as well but without biotin/avidin blocking. Slide 1 was treated with the 4 stage anti-Myc technique, and slide 2 was treated with the 3 stage mouse monoclonal technique. REFERENCES Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000 Jan 7;100(1):57-70. Baron, E. J. 1996. Classification. In S. Baron et al., eds. Barons Medical Microbiology, 4th edition. University of Texas Medical Branch. Gura, T. Therapeutic antibodies:Magic bullets hit the target. Nature 417, 584-586 (6 June 2002) Carter, P. Improving the efficacy of antibody-based cancer therapies Nature Reviews Cancer 1, 118-129 (November 2001) Sten Hammarstrà ¶m. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Seminars in Cancer Biology. Volume 9, Issue 2, April 1999, Pages 67-81 A. Mayer, K. A. Chester, et al. Taking engineered anti-CEA antibodies to the clinic. Journal of Immunological Methods. Volume 231, Issues 1-2, 10 December 1999, Pages 261-273 Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577-80. GBoxer, SStuart-Smith, et al. Radioimmunoluminography: a tool for relating tissue antigen concentration to clinical outcome. British Journal of Cancer (1999) 80, 922-926. Bhatia J, Sharma SK, et al. Catalytic activity of an in vivo tumor targeted anti-CEA scFv:carboxypeptidase G2 fusion protein. Int J Cancer. 2000 Feb 15;85(4):571-7. Mayer, A., et al. Radioimmunoguided Surgery in Colorectal Cancer Using a Genetically Engineered Anti-CEA Single-Chain Fv Antibody. Clinical Cancer Research May 2000 6; 1711. APPENDIX Appendix 1 Experimental Procedures (UCL Cancer MSc Lab Practical 2 Handout, 12-13Nov09) E. Coli growth curves E.coli culture transformed with pUC119 containing either His- or Myc- tagged scFv MFE-23 (Culture 1 2) were incubated overnight. The expression of pUC119 was controlled by the lac operon, which could be induced by either lactose or lactose analogue isopropyl-1-ß-D-thiogalactoside (IPTG). The vector also encoded for ampicillin resistance. 30 µl of the media was added to 15ml growth media with ampicillin selection and 0.05% glucose. Both tubes on loose caps were placed into a 37? shaker at 225rpm and time was taken as t=0. Optical density (OD) of the culture was measured with a spectrophotometer at 600nm from t=0 at a 30min interval until reading reached OD=0.9. 1mM IPTG was added to each culture and the tubes were incubated at 30? overnight with shaking. A negative control was set up using the same volume of growth media 2xYT with 1mM IPTG for overnight incubation. ELISA assay 100 µl of 10 µg/ml CEA antigen was applied to strips 1-6, A-H of the 96-well ELISA plate. 100 µl of PBS was applied to strips 7-12, A-H as negative controls. The plate was covered with plastic film and incubated for 1 hour at room temperature. Each well was rinsed 4 times with PBS and blocked with 200 µl 5% Marvel milk/PBS. The plate was covered and incubated overnight at 4?. The plate was washed with PBS 4 times after the overnight incubation. Overnight bacterial cultures and the negative control were centrifuged at 4000rpm for 20minutes to obtain the supernatant. 100 µl of each supernatant sample and negative control was added to the ELISA plate according to the format diagram as seen in Appendix 2. The covered plate was left at room temperature for an hour. The plate was then rinsed twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl of secondary antibodies rabbit anti-MFE23 polysera, mouse monoclonal anti-HIS tag (TetraHis, Qiagen) and mouse monoclonal anti-MYC (Sigma) with a concentration of 1:1000 in 1% blocking solution was added to the wells according to the format diagram: anti-His A1-12 and B1-12; anti-Myc C1-12 and D1-12; anti-MFE E1-12 and F1-12. The covered plate was left at room temperature for an hour. The plate was washed again twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl horseradish peroxidase (HRP) conjugated antibodies against the secondary antibodies 1:1000 goat anti-rabbit HRP (sigma) and 1:500 sheep anti-mouse HRP (Sigma) in 1% blocking solution was added to the corresponding wells. The covered plate was left at room temperature for an hour, and then rinsed twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl of OPD substrate buffer was added to each well to detect the presence of HRP, giving a yellow-orange product in the case of positive outcome. 100 µl of 4M HCl was added when the colour has developed, and OD was measured at 490nm with an automated plate reader. Immunohistochemistry assay Five glass slides, each containing 2 colonic adenocarcinoma, 1 normal colon and 1 normal liver tissue sections were removed from the freezer and air dried for 5minutes. Slides were fixed in acetone in 10minutes and rinsed in tap water for 2minutes. Slides were flooded with PBS and Avidin blocking solution was applied to slides 1-4 for 10 minutes. Slides 1-4 were rinsed in PBS and applied with Biotin blocking solution for 10minutes followed by PBS wash. PBS was removed from slide 1 and flooded with 1:20 normal horse serum in PBS for 15minutes. Slide 1 was drained and applied with bacterial supernatant containing Myc-tagged scFv MFE-23 antibodies for 45minutes. Slide 1 was then rinsed with PBS thrice over 10minutes, and 1:20 normal horse serum in PBS was added to slides 2-5 for 15minutes. 20 µg/ml of mouse monoclonal anti-Myc antibody (Qiagen) in PBS was added to slides 13 and left for 35minutes. 20 µg/ml of A5B7 anti-CEA antibody was added to slide 2 for 35minutes. Slides 4-5 were washed in PBS. Slides 1-3 were rinsed with PBS thrice over 10minutes. 1:200 biotinylated horse anti-mouse immunoglobulins in PBS with 5% normal human serum was added to all slides and incubated for 35minutes. All slides were rinsed with PBS thrice over 10minutes. The avidin biotin-peroxidase complexes reagent (ABC reagent) was added to all slides, followed by PBS wash 3 times over 10minutes. 0.03% of hydrogen peroxide in 1mg/ml 3,3,diaminobenzidene tetrahydrochloride solution was applied to all slides immediately and left for 5minutes. Slides were washed with tap water. All slides were stained in Harris haematoxylin for 40seconds, rinsed in tap water and left for 5minutes for colour to develop. Slides were then dehydrated through graded alcohols (70/95/100%), cleared in inhibisol and coverslip using DPX mountant. The slides were then ready for observations under the light microscope.