The Rationale behind Antibody Usage in Common Laboratory Assays - Essay Example

? The Rationale behind Antibody Usage in Common Laboratory Assays Such As ELISA, Immunofluorescence, Immunohistochemistry, and Western Blotting, their Relative Advantages and Disadvantages Student’s Name Professor’s Name Biology 08 November 2012 Table of Contents Table of Contents 1 Conclusion 15 List of figures Figure 1: ELISA…………………………………………………………………………………5 Figure 2: A representation of direct and indirect immunofluorescence…………………..9 Figure 3: Fluorescence overlap of phycoerythrin (PE) and (FITC) Fluorescein Isothiocyanate………………………………………………………………………………….10 Figure4: Principles of immunohistochemistry………………………………………………12 Figure 5: Steps involved in western blotting………………………………………………..14 The Rationale behind Antibody Usage in Common Laboratory Assays Such As ELISA, Immunofluorescence, Immunohistochemistry, and Western Blotting, their Relative Advantages and Disadvantages Introduction The immune system produces antibodies in reaction to an assortment of foreign substances as well as infections in the body. Many clinical laboratory tests incorporate antibodies because of their unique feature in identifying and singling out among very much interrelated antigens (Estridge & Reynolds 2011). Any test concerning an antibody-antigen reaction is referred to as a serological test (Kudesia 2006, p. 197). These tests make use of the reality that a good number of viral, bacterial, fungal, and parasitic illnesses bring forth accomplished antibody rejoinders. Therefore, identification of antibodies to infection agents in patients’ samples helps in the discovery of infections such as influenza, HIV, hepatitis, and rubella (Estridge & Reynolds 2011). There are a number of instances where it is not trouble-free to isolate the actual disease causing pathogens (for example syphilis and Toxoplasma). In such incidences, serological tests have been of utmost significance in making diagnosis (Kudesia 2006). Commercial microbe-specific antibodies detect antigens in patients’ specimens. Antigen-antibody rejoinders can also establish substances that are not part of the immune system, for example, hormone or drug levels, and even identification of a bacterial culture. Immunological tests make use of monoclonal or polyclonal antibodies. Antibodies of one class with specificity to only one epitope are called monoclonal antibodies. These antibodies are derived from a single cell line hence the name monoclonal. They are manufactured in the laboratories, making up the key reagents in most commercially available immunodiagnostic supplies. Polyclonal antibodies, on the other hand, are assortments of antibodies generated by many cell lines. These commercially available antibodies make use of a cloning process during their manufacture. The advantage of cloning antibodies is that it results in higher specificity in the antibodies. Both monoclonal and polyclonal antibodies can be produced from living organisms and cell cultures in the laboratory (Buchwalow & Bocker 2010). Scientists use mice, rats, and rabbits to clone antibodies. Antibody cloning is the production of antibodies from both living and non-living sources. The first step of monoclonal antibody production in rats involves injecting the rat with an antigen that elicits an immune response by production of antibody-forming cells. These antibody forming cells (B-cells) are isolated from the organism. The B-cells then fuse with tissue cultured tumor cells to form hybridomas (Buchwalow & Bocker 2010). The hybridomas then undergo screening for antibody production after which they are cloned into many hybridomas. The final step is the isolation of monoclonal antibodies from the hybridomas. Experience shows that monoclonal antibodies from rabbits exhibit more specificity than antibodies from rats and mice (Buchwalow & Bocker 2010). Measurement of immune responses following an infection makes use of the class of antibody formed by the reaction and the antibody characteristics. IgM and IgG are the most significant antibodies utilized in diagnosis of ailments. Serum measurement of IgA is also useful during such procedures. However, the use of IgA is practically more challenging and, therefore, is not used in routine laboratory diagnosis. Identification of the presence of antibody-antigen reactions involves observation for features such as agglutination and precipitation. Agglutination is the discernible clustering or aggregation of cells or fragments because of reaction with given antibodies. IgM exhibits the best outcomes in agglutination reactions due to its massive size and multivalent binding aptitude (Estridge & Reynolds 2011, p. 441). In most cases, agglutination indicates positive results whereas the absence of agglutination shows negative results. Precipitation, on the other hand, involves the establishment of an insoluble compound when a soluble antigen reacts with a given antibody. IgG responds best in precipitation reactions. Numerous immunological assays exist that are utilized in laboratory diagnostics. They include ELISA (Enzyme-linked Immunosorbent Assay), immunofluorescence, immunohistochemistry, western blotting, and immunomagnetic technology among many others. Enzyme-Linked Immunosorbent Assay ELISA is among the most prevalent serological tests in modern use. It involves the addition of an antigen to a hard surface, mostly polystyrene or polyvinyl shield. Incubation follows the addition of patient’s serum to give sufficient time for binding of the antibody to the surface. Washing the plate then follows to get rid of excess serum and non-specifically bound substances. Thereafter, “an antihuman immunoglobulin labeled by conjugation with an enzyme is then added” (Kudesia 2006, p. 200). Washing removes the surplus conjugate, adding an appropriate substrate reveals the existence of the attached labeled anti-human immunoglobulin. A coloured light-absorbing product reveals the occurrence of a chemical reaction. The strength of the colour is unequivocally relative to the quantity of antibody attached to the antigen. Figure 1: ELISA (ELISA. Causes, symptoms, treatment ELISA n.d.). Spectrophotometry quantifies the intensity of colour to give accurate measurements. However, the naked eye can also be used to measure this colour strength. Horseradish peroxidase (HRP), alkaline phosphatase, O-phenylene diamine, and p-nitrophenyl phosphatase are some of the enzyme-substrate in frequent utilization (Kudesia 2006). The solid phase is usually covered with an antibody that is specific to the antigen to be determined. Any antigen-antibody compound on the surface of the solid surface is detected by the addition of a second labeled antibody (secondary antibody). ELISAs may be categorized into several groups according to the sequence of the reactions. ELISA has several advantages over other serological techniques. It gives extremely high sensitivity because it requires only a small amount of sera (Leung & Gershwin). It also provides room for the examination of “multiple sera or multiple antigens on the same plate” (Leung & Gershwin p. 38). ELISA attains additional objectivity when compared to immunofluorescence because it utilizes spectrometric reading to sense antigen-antibody reactions. In addition, ELISA allows for partial automation of the pipetting station, something that is useful for a busy laboratory. According to Windt, et al. (1989), ELISA is both a qualitative and quantitative test and has the capability of distinguishing between different types of antibody classes. This property made it possible for them to detect secretory immunoglobulin A, which was not possible with other methods. They also argue that many scientists have found ELISA to be better than other immunological techniques. Among the most recent studies by Zoltewicz et al (2012), medical practitioners found a useful way of characterizing antibodies that identify glial fibrillary acidic protein (GFAP), a biomarker that is released into blood and the cerebrospinal fluid following traumatic brain injury (TBI). Detection of such proteins is crucial for the proper treatment and management of head trauma patients. This is because brain damage is among the leading causes of death and disability in the human populace. The study sought to determine the accuracy in terms of band width of two antibodies in a sandwich ELISA. The arrangement and schedule for the appearance of the biomarkers (glial fibrillary acidic protein) in bio fluids shows a possible molecular signal competent to define damage-linked strictures such as the extent of injury and results of the injury. As a consequence, there is immense predictive and analytical promise in developing tests that perfectly and exclusively quantify TBI-linked biomarkers particularly from blood specimens (Zoltewicz et. al 2012). The study realised that GFAP quantities in serum predicted mortality and an unflattering ending at a cut-off of 1.5 nanograms per millilitre. However, previous studies indicate that ELISA also has its shortcomings. Khan, Richardson, Warnock, and Lane (1984) established the unreliability of ELISA in the diagnosis of Aspergillus fumigatus, an intranasal infection in dogs. In their study, they obtained false positive findings in twenty seven healthy dogs. On that occasion, counterimmunoelectrophoresis proved more reliable. Windt et. al also found that ELISA correlates poorly with other immunological methods (1989). Immunofluorescence Fluorescence is the capacity of a substance to release light with no obvious delay when irradiated. Immunofluorescence is, therefore, the combination of fluorescence microscopy with the use of antibodies (coupled with chemically stable fluorochromes) to determine antigen-antibody reactions (Storch 2000). It is based on the pioneer efforts of Coons and Kaplan (Robinson, Sturgis, & Kumar n.d.). Immunofluorescence plays a key role in applications such as the study of cells in suspension, tissues, beads, cultured cells and microarrays in the finding of exact proteins. FITC (Fluorescein Isothiocyanate) is the regularly utilized fluorescent dye in labeling antibodies. It emits yellow-green light at wavelengths of about 500 to 550 nanometers. Secondary fluorescence is accomplished synthetically through using fluorescent substances, whereas primary fluorescence is the natural fluorescence emission from untreated materials. Immunofluorescence encompasses three different approaches namely the direct method, indirect method, and anti-complement method (Storch 2000). In the direct approach, the sample under investigation is treated with fluorescently labeled antibodies (the primary antibody). Fluorescence microscopy identifies only those cells that are linked to the labeled antibody. This method is, however, rarely used. According to Robinson, Sturgis, and Kumar, direct immunofluorescence provides brief staining durations (n.d.). It is also possible to identify multiple antibodies in the same species through simple dual and even triple labeling processes. However, indirect labeling has the disadvantage of being expensive and less flexibility. Direct labeling poses problems in instances where commercially labeled direct conjugates are not available. Indirect labeling reveals humoral antibodies aimed against recognized cell and tissue proteins and involves utilization of as labeled antibody (secondary antibody) to spot another antibody (primary antibody), thereby making a sandwich (Estridge & Reynolds 2011). This procedure offers higher sensitivity compared to direct immunofluorescence. The high sensitivity is as a result of magnification of the signal since many secondary antibodies can bind to the primary antibody as indicated in the below illustration. This approach is also comparatively economical because Commercial secondary antibodies are relatively reasonably-priced, available in a range of colours, and quality controlled (Robinson, Sturgis, & Kumar n.d.). The disadvantage of indirect labeling is that there is the possibility of cross-reactivity. It is, therefore, imperative to look for primary antibodies that are not brought up in similar species, or using different isotypes during multi-labeling tests. Figure 2: A representation of direct and indirect immunofluorescence (Robinson, Sturgis, and Kumar n.d.). The anti-complement system depends on the attachment of the component of the complement system in the course of “antigen-antibody complex formation” (Storch 2000, p. 3). This method is useful for the demonstration of antigen-antibody reactions that occur in vivo, and both direct and indirect labeling is possible. Immunofluorescence as a serological technique has several limitations. One such limitation is photobleaching. According to Robinson, Sturgis, and Kumar, photobleaching is the damaging of a fluorophore due to photochemical effect (n.d.). Fluorescence excitation produces a reactive oxygen atom in the sample as a by-product resulting in photobleaching. The exact is unknown; however, scientists think that the prime causal machinery is light sensitization of singlet oxygen by the dye in the triple-excited state (n.d.). Lessening length and potency of the excitation light, decreasing the accessibility of singlet oxygen (1O2) by including singlet oxygen foragers may help curtail photobleaching. In addition, utilizing a low intensity of fluorochromes with high quantum effectiveness also lessens photobleaching. Autofluorescence is another problem in immunofluorescence. Flavin coenzymes and reduced pyridine nucleotides bring about autofluorescence, which makes it difficult to distinguish fluorescent probes in tissues and cells. Fixing samples with aldehydes also brings about autofluorescence. Washing cells with 0.1% sodium borohydride in phosphate-buffered saline solution before incubation with antibodies alleviates this problem (Robinson, Sturgis, & Kumar n.d.). Functioning of the detection apparatus (proper calibration and settings), antibody specificity, and preparation of the samples are other factors that determine the accuracy of immunofluorescence. Emission signals can overlap when dealing with many colours leading to fluorescence overlap. It is, therefore, imperative to do away with the overlapping signal to avoid giving false results for that colour. Figure 3: Fluorescence overlap of phycoerythrin (PE) and (FITC) Fluorescein Isothiocyanate (Robinson, Sturgis, & Kumar n.d.). Immunohistochemistry Immunohistochemistry is a technique that utilizes antibodies to spot and envisage antigens in cells and tissues. Tissue fixation is a critical procedure before any immunohistochemical procedure because it helps to maintain the integrity of tissues and antigens by forestalling autolysis. The choice of fixation technique depends on the qualities of the tissue, antigen, and antibody (Immunohistochemistry microimaging techniques, n.d.). Physical or chemical methods can fix tissues before the analysis. Physical fixation involves freezing the tissues in liquid nitrogen and cutting the frozen tissue thereafter. Chemical fixation, on the other hand, involves the utilization of cross-linking, precipitating, or oxidizing agents. The tissue is immersed in any of these agents for a specified duration according to the reagent. Removal of water then follows and is carried out by embedding the tissue in wax or resin. The resin or wax-embedded regions are then cut off leaving the tissue ready for analysis. The second step during analysis involves washing the tissue in physiological buffers or balanced salt solutions. The third step, which is extremely significant, entails blocking of non-specific binding to the tissues by incubation in normal serum. After this, the tissue is incubated with the antibody that is specific to the antigen in question, washed in physiological buffer, and detected using a detection system. The labeled specimen is finally mounted on a slide and examined microscopically (Immunohistochemistry microimaging techniques n.d.). Figure 4: Principles of immunohistochemistry (Immunohistochemistry microimaging techniques, n.d.). Immunohistochemical detection methods employ immunofluorescence (observation of fluorescence from fluorochrome-conjugated secondary antibody) and enzymatic techniques (enzymatic conversion of a soluble substrate into an insoluble and colourful reaction product) (Immunohistochemistry microimaging techniques n.d.). Immunohistochemistry enables the assessment of expression of different receptor activators in a myriad of diseases, for example, in periodontitis, which is a disease-caused inflammatory disorder. Giannopoulou, Martinelli-Klay, and Lombardi, through immunohistochemistry identified that RANKL was not “expressed in the oral and periodontal pocket epithelium” (2012, p. 629). OPG was also not expressed in the entire epithelium. However, RANK was expressed mainly in the basal and suprabasal layer of oral and periodontal pocket epithelium” (Giannopoulou, Martinelli-Klay, & Lombardi 2012, p. 629). The main advantage of immunohistochemistry is that it provides for the actual examination of a living tissue and, therefore, gives higher chances of accurate diagnostics. However, its limitation is that fixation is a basic requirement. Fixing kills living tissues and so a tissue must first be isolated from the living organism before the assay can be performed. Western Blotting Western blotting, also known as immunoblotting is a method that combines antibody-labeled tests and electrophoresis (Estridge & Reynolds 2011). Several methods can label the secondary antibodies employed in immunoblotting, for example, fluorescent dyes, enzymes, and chemiluminescent molecules (Estridge & Reynolds 2011). Separation of antigens from a nuclear extract happens according to molecular weights using SDS-PAGE (sodium dodecyl sulphate) electrophoresis. The separated antigens are afterwards moved to nitrocellulose paper. This transfer of antigens to nitrocellulose paper is what is referred to a blotting. The nitrocellulose paper containing the antigens is reduced to single narrow pieces and incubated with the patient’s serum (Misbah 2006). Enzyme-labeled anti-human immunoglobulin detects the attached antibodies on the nitrocellulose paper by further incubation. Figure 5: Steps involved in western blotting (GenScript IP Western Blot Kit Overview, n.d.). The main advantage of western blotting is its specificity. Therefore, it is not commonly used in clinical immunology laboratories as it is set aside for the examination of patients’ samples yielding discrepant outcomes by the regular tests. A valid example is in patients showing convincing clinical signs of lupus in whom standard tests do not succeed in discovering antibodies to DNA and ENA (Misbah 2006). Enzyme-linked immunosorbent assay (ELISA) is the best serological test after taking a careful look at the strengths and weaknesses of each of the serological tests. This is because it gives both qualitative and quantitative results. This information is much more valuable in making an accurate and informed decision on the best treatment option for a patient or the next best course of action if the assay does not involve medical diagnosis. Conclusion All the mentioned serological tests discuss making use of antigen-antibody reactions in determining the presence or absence of a given condition in the patients’ samples. The basic principle in all the methodologies is the same, what changes in each methodology are only small details in the sequence of steps, or method of visualizing the final results. In addition, all these methods are not independent of each other since some studies require a combination of different immunological assays to come up with a comprehensive result. For example, the characterization of antibodies to detect GFAP in distressing brain damage requires the use of several immunological tests such as immunoblotting, immunoprecipitation, and ELISA (Zoltewicz et. al 2012). Immunological techniques are vital in establishing various life-threatening conditions, and even serve as guides to providing proper medical treatment to patients. Hence, it is extremely significant that all antibodies used in the assays provide the highest degree of accuracy. This enables precise diagnosis and consequently precise treatment. The overall result is the ability to salvage as many lives as possible. This is why it is imperative for further research to be done in order to do away with errors and eliminate most shortcomings of each immunological assay. References Buchwalow, I. B. & Bocker, W 2010, Immunohistochemistry: Basics and methods, Springer, Heidelberg, Germany. ELISA. Causes, symptoms, treatment ELISA, n.d., viewed 14 November 2012, http://drugline.org/ail/pathography/1519/. Estridge, B. H. & Reynolds, A.P 2011, Basic clinical laboratory techniques, 6th edn, Cengage Learning, New York, USA. GenScript IP Western Blot Kit Overview, n.d., viewed 14 November 2012, http://www.ipwestern.biz/. Giannopoulou, C., Martinelli-Klay C.P., & Lombardi, T 2012, “Immunohistochemical expression of RANKL, RANK and OPG in gingival tissue of patients with periodontitis,” Acta Odontologica Scandinavica vol.2012, no.70 pp. 629-634. Immunohistochemistry microimaging techniques, n.d., viewed 08 November 2012, . Khan, Z. U., Richardson, M. D., Warnock, D. W., & Lane, J. G 2007, “Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Aspergillus fumigates intranasal infection of the dog,” Journal of Medical and Vetinary Mycology vol. 1984, no. 22, pp. 251-254. Kudesia, G 2006, “Serological tests in virology,” in John Crocker and David Burnett (eds), The science of laboratory diagnosis, John Wiley & Sons, Hoboken, NJ, USA. Laboratory Methods in Endocrinology, n.d., viewed November 14 2012, http://www.trinity.edu/lespey/biol3449/exercises/Methods.html. Leung, P. S. C. & Gershwin, M. E “Native autoantigens versus recombinant autoantigens,”.in Shoenfeld, Y., Gershwin, M. E., & Meroni, P. L(eds), Autoantibodies, 2nd edn, Elsevier, Burlington, MA, USA. Misbah, S. A 2006, “Serological tests in virology,” in John Crocker and David Burnett (eds), The science of laboratory diagnosis, John Wiley & Sons, Hoboken, NJ, USA. Robinson, P. J., Sturgis, J., & Kumar, G. L n.d., Immunofluorescence, viewed 07 November 2012, . Storch, B. W 2000, Immunofluorescence in clinical immunology: A primer and atlas, Springer, Berlin. Windt, M. L., Bouic, P. J. D., Lombard, C. J. Menkveld, R., & Kruger T. F 1989, “Antisperm antibody tests: Traditional methods compared to ELISA.” Archives of Andrology, vol.1983 no. 23 pp. 139-145. Zoltewicz, J. S., Scharf, D., Yang, B., Chawla, A., Newsom, J.K., & Fang, L 2012, Characterization of antibodies that detect human GFAP after traumatic brain injury, Biomarker Insights, vol. 2012 no.7 pp. 71–79. Read More