Clinical Chemistry - Immunoassay - Lab Report Example

CLINICAL IMMUNOLOGY LAB REPORT Introduction Immunoassay involve bio-analytical technique whereby the quantification of the analyte relies on the reaction of the analyte which, is always an antigen on the antibody (Macario, & Macario 2004). History of immunoassays dates back in the mid twentieth century. Dr. Solomon Berson together with his partner Dr. Rosalyn Yalow performed investigative studies concerning insulin breakdown in patients with diabetes at the Bronx veterans Administration Hospital in 1950 (Zigova 2002). The doctors determined that globulins that bind to insulin among the patients who were insulin-treated were in fact antibodies. After the first development of immunoassays, the techniques have undergone a series of developments that have also led to the advancement of the technologies used (Wong & Tse 2009). The techniques have been applied in the field of clinical immunology and other numerous facets of clinical laboratory. In fact, the techniques of immunoassay systems has enhanced diagnostic and well as research methodologies greatly (Asai 2003). Many drug testing facilities use immunoassays as the conventional method drug testing technique due to its ease in application and the extensive automation. Types of ELISA Competitive ELISA Is an ordinary ELISA though has a slight modification to the common protocols. In this scenario, a biotinated substance is introduced to compete with the antibody, antigen to bind to the previously included antigen, antibody for the duration of the reaction. The aim of introduction of the competitor substance is to avert pointless binding of antibody or else the antigen and ensure that the binding only ensures a greater affinity amid antigen or antibody rather than their mere inclusion. Monoclonal ELISA The antibodies signify a solitary B lymphocyte producing antibodies to a single specific epitope. An easier isolation of B-cells is possible from the spleen and lymphnodes of vaccinated organisms. Nonetheless, such cells have restricted lifetime and can only involve in a limited division series, Hayflick limit. As a result, the restriction directly limits the culture of B- cells. All antibodies have to meet a sole characteristics of being available in abundant quantities in order to be useful in research. Because of Hayflick limit, abundance availability might not be probable through the use of B-cells cultured in vitro because they would stop dividing at long last leading to elimination of dying out (Asai 2003). A research performed in 1975 by Milstein and Kohler led to the creation of a technique with the abpility to combine lymphocytes the heteromyleoma cells through the use of poly ethylglycol (PEG) to disintegrate cell membranes thus permitting the mixture of the genetic components from the constituent types of cells to form a hybridoma cell (Peng & Zhao 2009). The hybridoma cell is special as it contains the features of both the constituting cells and is an immortal cell that is capable of producing an antibody. Because the hybridoma is a result of a single B- cell and a single heteromyeloma cell as well, the culture has only one antibody in the supernatant that becomes the monoclonal antibody when purified. This technique permits researchers to extract and refine one antibody from the composite mixture of antibodies available in the in vivopolyclonal reaction (Asai 2003). Such cell line, once made stable through one cell cloning, can be dried in ice and kept indeterminately in liquid nitrogen, permitting the antibody to be generated in vitro, in the necessary greater amounts as demanded in research (Zigova 2002). These antibodies can be leveled against several targets. Particular features of can be recognized and selected for instance, the sensitivity demands and cross reactivity extents can be determined besides the screening of monoclonal antibodies to recognize any cell lines manifesting the features (Wong & Tse 2009). The antibodies in this category can also be produced to cross react with a section of molecules for instance the tricyclic anti-depressants possesses the same overall conformation with replacements of divergent constituents into the cyclic conformation. In essence, this is very crucial in clinical drug identification when many probable combinations for drug requires to be tested in a patient (Wild 2007). Polyclonal ELISA Polyclonal are the large quantity of antibodies generated with diverse specialisms together with epitope affinities as well. The polyclonal antibodies result because the immune response targeting antigen demands the activation of several B- cells and which targets a particular epitope on an antigen in unison (Macario, & Macario 2004). The antibodies are purified from the serum of vaccinated organisms in which the targeted antigen stimulates the B-lymphocytes to release a wide range of immunoglobulins specific to the respective antigen for production reasons. The target is to generate antibodies with a high concentration of the highest affinity (Macario, & Macario 2004). Presently, the polyclonal antibodies are applied vastly in research operations in major biological fields especially the enzyme linked immunosorbent assays (ELISA), immunoprecipitation, diagnosis of conditions and the western blots as well. The antibodies of this category can also be applied at the second stage of the sandwich assays to identify antigens (Asai 2003). The polyclonal antibodies in large amounts are comparably fast and inexpensive as compared to other antibodies. The antibodies are non-specific and are able to identify many epitopes on every single antigen (Wild 2005). Therefore, the feature can assist in increasing the signal generated by the protein aimed since the antibody will bind to more than a single epitope (Wong & Tse 2009). The polyclonal antibodies manifest least sensitivity to alterations to changes in antigens as compared to other antigens in the category such as monoclonal antibodies. Because of the multiple epitopes, they manifest more vigorous detection capabilities. Sandwich ELISA The sandwich ELISA determines the quantity of antigen amid two antibody layers. The antigen to be evaluated has to comprise of a minimum of two sites of antigen that have the capacity to bind to the antibody because the sandwich consists of two antibodies acting on it (Wild 2005). Sandwich ELISA systems are versatile and can accept any of the monoclonal or polyclonal antibodies as the capture and detection antibodies. The Monoclonal antibodies detect one epitope which, permits refined detection as well as estimation of small variations in antigen (Ritter & Ladyman 2004). The polyclonal is normally applied as the capture antibody to attract the maximum quantity of the antigen as probable. Sandwich ELISA is advantageous in its application since the purification of the sample before analysis is not necessary (Wild 2007). As comparted to the direct and indirect types of ELISA, sandwich ELISA is sensitive up to two to five times (Wong & Tse 2009). In clinical immunology, it is critical to understand that procedures in Sandwich ELISA can be challenging and confirmed match paired antibodies should be employed. The involvement of match paired antibodies makes sure that antibodies recognize variant epitopes on the target protein without interfering with alternate antibody binding. Because of this observation antibodies can only be guaranteed after confirmatory tests for their specificity for sandwich ELIZA (Arneson & Brickell 2007). Discussion The objectives of the experiment were achieved through two series of practicals, 1 and 2. The first session of the experiment was set out to determine the optimum dilution of monoclonal mouse anti-rabbit IgG and polyclonal goat anti-rabbit IgG through by the use of sandwich ELISA. The mouse anti-rabbit IgG was used in concentrations ranging from 2000ng to 31ng/ml whilst the mouse anti-rabbit IgG applied concentrations ranging from 2000ng to 32ng/ml. In both situations, the experiment also involved the concentration of 0ng/ml. Moreover, the concentration of 0ng/ml worked as blank. Increasing the working dilution and the antigen concentration leads to an increase in absorbance throughout the exercise. At a concentration of 0ng/ml, the absorbance is zero. The spectrophotometer applies the principle of absorption of light by the molecules in the analyte reducing the intensity of beam (Arneson & Brickell 2007). The resultant beam is digitized and read on the display screen. The lower the intensity leads to the higher reading of absorbance. The polyclonal antibodies from goat immune-affinity refined utilizing conforming restrained antigens and conjugated to the enzyme. The Anti-Rabbit IgG antibodies bind to both weighty and light chains for all the subclasses of IgG. As with all antibodies, in certain applications some species-dependent antigen-dependent cross-reactivity might be observed. The outcomes are provided as the 1mg/ml solutions. The starting working dilution of 1:2000 is proposed for most ELISA uses (Peng & Zhao 2009). The secondary antibody’s optimum concentration of relies on the solicitation and will require to be determined in an empirical manner. The rabbit IgG was applied as an antigen in various dilutions as outlined, the readings taken and recorded in the tables shown. The best curves were obtained from the results and displayed in the results as well on the IgG concentrations against the absorbance. There is a realization that the resultant curves from 1/2000 and 1/4000 dilutions utilized more antibodies. Monoclonal antibodies are more expensive and their affordability is limited. Being that the antibodies are obtained commercially, it is not allowed due to the higher costs involved. Alternate curves shown from the dilutions such as 1/16000, 1/32000 together with 1/64000 never manifested any distinct variation and no reading amongst them can be selected as optimal dilutions. The second practical incorporated the display of the absorbance results from the dilution of Goat anti-rabbit IgG antibodies as demonstrated in the second table. The dilutions ranging from 1/16000 to 1/64000 depicted a linear curve and no optimum value could not be deduced. However, the remaining three values ranging from 1/2000 to 1/8000 shown a steady increase with the first two points (1/2000 and 1/4000) rose to a particular extent until the maximum point of rabit IgG to the stationary region. Thus, it is possible to consider the dilution of 1/4000 as the optimum according to the observations. The standard curve is one of the best means f determini9ng concentrations of unknown substances. In the second part of the practical, there was need to determine the concentration of substances X and Y. it is possible to identify the unknown concentrations of substances by drawing straight lines from points of known concentrations on the curve on the rabbit IgG by the use of Sandwich ELISA. According to the graph, estimated to value of X = 514.039 ng and the concentration of Y = 85.858 ng. ELISA is the best means for determining accurate results that can lead to definite evaluations and determination of diseases since they are less expensive. A number of errors might have hampered the accuracy of results ranging from environmental errors such as temperature, disparity in reagent mixing and inappropriate cleaning of wells among others. Conclusion The optimum dilution was determined to be at 1/4000. The concentrations of X and Y were determined by the Rabbit IgG to be 514.039 ng and 85.858 ng/ml respectively. Therefore, the experiment attained its objectives successfully. Again, it is critical to understand the essence of optimum dilution of antibodies to use the least quantities of antibodies as they are expensive. Bibliography Arneson, W., & Brickell, J. 2007. Clinical chemistry a laboratory perspective. Philadelphia, F.A. Davis Co. http://search.ebscohost.com/login.aspx?direct=true&scope=site&db=nlebk&db=nlabk&A N=194356. Asai, D. J. 2003. Antibodies in cell biology. San Diego, Academic Press. http://www.sciencedirect.com/science/book/9780125641371. Diamandis, E. P., & Christopoulos, T. K. 2006. Immunoassay. San Diego, Academic Press. http://site.ebrary.com/id/10191442. Macario, A. J. L., & Conway De Macario, E. 2014. Monoclonal Antibodies Against Bacteria Volume II. Burlington, Elsevier Science. http://public.eblib.com/choice/publicfullrecord.aspx?p=1875192. Peng, S., & Zhao, M. 2009. Pharmaceutical Bioassays Methods and Applications. Hoboken, John Wiley & Sons. http://www.123library.org/book_details/?id=4526. Ritter, M. A., & Ladyman, H. M. 2004. Monoclonal antibodies: production, engineering, and clinical application. Cambridge, Cambridge University Press. Wild, D. 2005. The immunoassay handbook. Amsterdam, Elsevier. Wild, D. 2007. The immunoassay handbook. Amsterdam, Elsevier. Wong, R. C., & Tse, H. Y. 2009. Lateral flow immunoassay. New York, NY, Springer. Zigova, T. 2002. Neural stem cells: methods and protocols. Totowa, NJ, Humana Press. Read More