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Introduction and Aims
Salmonella is the scientific name of a bacteria and is a known foodborne pathogen and a leading cause of gastroenteritis in human beings. Salmonella can be categorized into more than 2,500 serotypes based on O and H antigens (Cabello et al. 2012). Therefore, three methods are used to detect H antigen in Salmonella typhimurium, in this practical sonication, the Bradford protein assay and the ELISA methods are described. The concept of sonication procedure involves unbounding and releasing the components matter. In the medical laboratory, a sonication method was used to fracture the cell wall of the bacteria to discharge its components and its mainly conducted under high-frequency sound.
The quantity of protein in a solution can easily be determined using its absorbance that is detected using a spectrophotometer. Bradford assay is a type of primary assay base on light solution absorbance light. The process of determining the number of proteins in a solution entails the combination of protein solution and Binding Reagent Dye to determine the concentration gradient. Once the solution is mixed with protein, the dye becomes unprotonated. Therefore, a calibration curve of absorbance against protein concentration forms Bradford's protein assay is utilized in detection of protein concentration. Enzyme-linked immunosorbent assay ELISA is a technique that is plate-based and is designed for detection and quantification of peptides, antibodies, proteins, and hormones.
The immune- chemistry is mainly applied in the medical laboratories to give easy, sensitive and fast methods that can be automated for everyday analysis (Davis, 2018). The enzyme-linked immune-sorbent assays (ELISA) is mainly used assay which is based the principle of Antigen and Antibody which bind with photometric detections (Hosseini et al. 2018). In the determination of antibody titer, indirect ELISA test is considered most appropriate in the determination of the generated antisera. ELISA method is most efficient and more economical to be used in the medical laboratories since it does not require tedious sample preparation instruments hence, it gives a rapid detection of antibodies or antigens which are associated with certain diseases, hormones or biomarkers in specific. In an ELISA test, it is a requirement that an antigen must get immobilized and complexes with an antibody linked to an enzyme. Thus, the results of ELISA procedures are mainly characterized by colored end products which correlate to the analyte amount presents in the original sample.
More so, the ELISA methods we are used in the medical labs have the potential to detect proteins of less than a nanogram (10-9g). The concept of ELISA can apply either monoclonal or polyclonal antibodies. However, it was found that monoclonal antibodies produce more reliable results in a lab practical. Notably, there are some types of ELISA which include the indirect ELISA that is commonly applied to detect the availability of antibody and therefore it is the basis of many tests such as HIV test and the Sandwich ELISA, in this type of ELISA, the antigen can be quantified and detected. More so, there is direct and competitive ELISA.
The main aim of this experiment of immune was to find out the concentration of antibody which is required for the detection of flagellar H-antigen of Salmonella Typhimurium to be noticed easily using indirect ELISA. Escherichia Coli was being used as a negative control experiment because it does not give the flagellar antigen. Also, the Bradford assay, which has shown a rapid and simple method to quantify proteins in the cell lysates was applied to quantify the number of proteins in the bacterial cell lysates. The Bradford assay was mainly used to measure the exact protein concentrations.
Discussion and Final Conclusion
In the first day of the practical experiment, three crucial steps were followed. The first step was the isolation of the bacteria protein as H-antigen by the sonication concept. The second step was measuring the protein concentration in the solution by the use of Bradford assay. Finally. Coating of the microtiter plate using the H-antigen was done.
In the practical, experiment, the concentration of protein in a solution was measured and, therefore, Bovine Serum Album (BSA) was combined with the samples to reading on the absorbance instrument was 595 nm. The results obtained were fed to Excel to develop a scattered plot that illustrates the standard curves for the first concentration of 0.3153 mg/ml. Additionally, the equation meant to indicate the dilution of H-antigen is 47.8 ml out of the entire sample of 3 ml that was used to compute the quantity of protein in the total sample.
The main reason of the H-antigen volume was to coat the microtiter plate which was used in the indirect ELISA type (Bruins et al. 2015). More so, the coating of H-antigen was conducted in to add to the secondary and primary antibody. The addition of 450nm of TMB substrate was basically done to dye the bound antigen-antibody thus, increases its visibility.
In the results of the experiment, nothing was detected for Shigella antisera, but salmonella pathogen indicated a binding reaction with the salmonella antisera. From the results of this practical, it was demonstrated that the coated samples which contain H-antigen showed a total binding when it was added with salmonella antisera with specific H-antigen antibody.
Consequently, the result showed that the addition of either secondary antibody or anti-antibody made this so sensitive and fast that it was ready to bind easily with the entire complex yielding a titer of 1.5ml. It was found that E. coli lack H-antigen and therefore, the high elevation of the results indicated that there is a cross-reaction between O and H antigens.
In conclusion, the results show that the coated samples which entail H-antigen showed a whole binding once it was added with salmonella antisera which contains specific H- antigen antibody. It is true that there is a homology epitope that exists between both antigens. Also, nothing was detected for Shigella antisera. Thus, E.coli was used as the negative control experiment. The most reliable test to be employed is the indirect and sandwich ELISA.
Bruins, S. I. D., Bremer, M. G. E. G., van, F. K. I., & Hamer, R. J. (2015). Evaluating the Performance of Gluten ELISA Test Kits: The Numbers Do Not Tell the Tale. (Cereal chemistry.)
Cabello, F., Hormaeche, C., Mastroeni, P., & Bonina, L. (2012). Biology of Salmonella. Boston, MA: Springer US
Davis, D. M. (2018). Beautiful Cure: The New Science of Immunity and the Dawn of a Health Revolution.
Hosseini, S., Vazquez-Villegas, P., Rito-Palomares, M., & Martinez-Chapa, S. O. (2018). Enzyme-linked immunosorbent assay (ELISA): From A to Z.
In Santambrogio, L. (2015). Biomaterials in regenerative medicine and the immune system.
New protein techniques. (2014). Place of publication not identified: Humana.
Organisation for Economic Co-operation and Development. (2010). Test No. 442B: Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA. Paris: OECD Publishing.
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