Abstract
The aim of the experiment is to plot the calibration curve for deteremination of antigen 4D5 by performing ELISA experiment. The primary antibody was blocked by bovine serum albumin, then it reacted with IgG (secondary antibody). Then, the system was washed with carbonate, blocking and substrate buffers. Three replicate experiments were performed, and the experiment was performed with the serial dilutions. By measuring the optical density of the diluted solutions, the calibration line was obtained and the region with the linear dependence “concentration – optical density” was determined. The callibaraion line can be used for the practical tasks: for detecting viruses, measuring antibodies level, and detecting hormonal changes.
Introduction
Imunoassay is a bioanalytical method of quantitative determination of analyte concentration, which is based on the reaction of antigen (analyte) and antibody. Imunoassay is a convenient method for measuring antibody levels, for viruses detection, and tracking hormonal changes. From the practival point of view, the method is used for allergies and vaccines research, for detection hepatitis, veneral diseases, HIV, at drug testing. The practical and scientific significance of the methods is caused by the specificity, sensitivity, and the wide range of analytes from the biological systems (Darwish, 219).
Enzyme-linked immunosirbent assay (ELISA) principles were derived from radioimunoassay in 1960 by Yalow and Berson. ELISA uses antibodies and enzyme-mediated color change to detect the presence of of proteins, peptides, hormones antigens or antibodies (Aydin, 6). The various subcategories of ELISA have been developed: "indirect" or "sandwich" methods for determination of antibody or antigen at ultra low concentration (Gan & Patel, 1).
Imunoassay
Figures 1-4 illustrate typical immunoassay formats.
The sample of primary antibody is added to the microtiter plate, where it adsorbs on the surface. The blocking agent is added to the solution to deactivate the sites of the plastic, which were not used by antibodies. The secondary antibody (conjugated) is added, which binds with the primary antibody. The substrate solution is introduced, and the solution often changes color. The intensity of the color depends on the concentration of the primary antibody (Price & Newman, 80).
Calibration curve is meant to estimate the concentration of biocomponent basing on the signal generated by the solution of the certain concentration. The solutions with the known concentrations of component are analyzed, and the curve concentration versus sighal is plotted. Basing on the signal produced by the solution with unknown concentration and using the callibration line, the concentration of the component can be determined. Figure 2 illustrates the calibration curve.
Figure 2: Theoretical callibration curve
The calibration curve has three regions:
The region of the low concentrations of biocomponent, where the signal is stable at several concentration values. This region is not suitable for calibration.
The region where the concentration is directly proportional to the signal. This region is used for calibration.
The region where the signal does not change with the increase of concentration, and therefore it is not suitable for calibration (Moo-Young).
Methods of Immobilization for ELISA
Biomolecules immobilization refers to decrease of biomolecule mobility. There are several methods, based on various properties of the biomolecules and other agents, which can be introduced to the solution. Adsorption is based on the decrease of surface tension of adsorbent by joining biomolecules. Encapsulations refers to inclusion of the biomolecule into the membrane, which is non-permeable for the boimolecules. When the biomolecule is trapped by insoluble matrices (calcium alginate), the immobilization is called entrapment. Covalent bonding is immobilization on surface by formation of covalent bond (reaction with the surface components), and this is interaction is typically irreversible. Cross-linkage is formation of covalent bonds betwen the biomolecule to create a matrix (Wild 284). Figure 3 illustrates the methods of biomolecules immobilization.
The serial dilution is used to create a series of concentrations prepared under the same conditions and from the same stock solution, which differ proportionally from one another. When analytical signals are obtained from this solution, they are used to plot the concentration - signal plot. The role of serial dilution is in generation of different concentrations with the certain level of precision (Danzer, 69).
The ELISA samples and standards were realized in replicates to minimize the random instrumental errors. When 2-3 replicates are analyzed , the repeated measurements compensate the random errors (Danzer, 72).
Results
Figure 4: Photo of the serial dilutions used for calibration.
Figure 5: Experimental calibration curve.
Discussion
The set of solutions resulting in serial dilution is presented in Figure 3. It can be seen that the solutions significantly differ in colour: there are solutions that are nearly transparent, and there are solutions with the distinct yellow colour. The concentrated solutions are yellow, and the less the concentration of primary antibody, the more transparent the solution is.
Three replicates are performed for each value of concentration. This is performed to ensure the accuracy of the experiment and to exclude the systematic errors. The replicate samples allow to compensate the random errors, and if one of the replicates significantly differ from the others, it is a sign of the systematic error. In this case, either the one more measurement is required, or this value has to be excluded from the measurements (Mager, 216).
The experimental measurements for absorbace are presented in Table 1. The replicate measurements indicate that there is no significant variation, and the obtained values are close. The average values were calculated and used for graph plotting. The calibration curve is presented in Figure 4.
As it is apparent from the calibration curve that there are three regions. Although the region 1 is not clearly seen, the analysis of the table values of trials 8-11 indicates that there is no significant change in signal with the concentration increase. Even more, the lowest concentration (trial 11) exhibits signal 0.126, which is higher than the signal for trial 7. The detailed analysis of the replicates of the trial 11 indicate that the absorbance values observed are not consistend with the previously obtained values. This is a sign that the dilution factor is too high, and concentration is below the limit of detection and thus the determination is not reliable and cannot be used for calibration. Therefore, the trials 8-11 cannot be used for calibration (Deshpande, 404).
The second region is represented by trials 3-7. In this region, the concentration is related to the signal: the higher the concentration, the greater the signal. This region can be used for calibration, according to the theoretical data presented in Wild (44).
The region 3 is represented by trial 2, and the linear relation of the concentration and signal is violated, therefore this point should be excluded from the data points used for the analysis.
Hence, the calibration line, which can be used to determine the concentration of the primary antibody sample should be plotted using the trials 3-7. Figure 6 illustrates the calibration line.
Figure 6: Experimental callibration line.
Using the equation of the caibtraion line, the concentration can be determined:
Concentration = (Optical Density – 0.1564) / 0.0981.
This equation is reliable since the R2-value is close to 1 (R2 = 0.9754), and this indicates that the equation perfectly describes the concentration – optical density dependence. The R2-value indicates that the variation in optical density is 97.5% caused by concentration change.
Although the calibration is reliable and accurate, it should be noted that the values of optical density are subject to variation. This is illustrated by the difference in the replicate measurements. Hence, the calibration line is valid only for one set of measurements, namely for one experiment. If the experiment is performed the next day, the new dilutions and the new calibration should be performed. In this case, the highest accuracy of the concentration determination is guaranteed (Law, 122).
Conclusions
The lab assignment was dedicated to realization on practice the ELISA method of antigen concentration determination. For this, the series of dilutions of Antibody 4D5 were prepared and processed according to ELISA procedure. The following laboratory operations were practiced: step and serial dilutions, incubation, washing, blocking, and substrate treatment. The principle of trial measurements for accuracy was tested experimentally. The experimental results were processed using MS Excel, and the data were used to plot the calibration curve.
The calibration curve fully corresponds to the theoretical curve presented in the literature. It was analysed and the region suitable for quantification of antigen was determined. The quality of calibration curve indicates that all the laboratory operations were appropriately performed and there were no systematic errors.
In this way, the skills on biochemical laboratory practices, data processing and analysis were trained. This improved understanding of imunoassays and importance of ELISA method.
References
Aydin, S. (2015). A Short History, Principles, and Types of ELISA, and Our Laboratory Experience with Peptide/Protein Analyses Using ELISA. Peptides, 72, pp. 4-15.
Danzer, K. (2007). Analytical chemistry: theoretical and metrological fundamentals. Berlin, Springer.
Darwish, I. A. (2006). Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. Interbational Journal of Biomedical Science, 2(3), pp. 217–235.
Deshpande, S. S. (1996). Enzyme immunoassays: from concept to product development. New York, Chapman & Hall.
Diamandis, E. P. and Christopoulos, T. K. (1996). Immunoassay. San Diego, Academic Press.
Gan, S. D. and Patel, K. R. (2013). Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. Journal of Investigative Dermatology, 133, pp. 1–3.
Law, B. (1996). Immunoassay: a practical guide. London, Taylor & Francis.
Price, C. P. and Newman, D. J. (2014). Principles and practice of immunoassay. Palgrave Macmillan.
Moo-Young, M. (2011). Comprehensive biotechnology. Amsterdam, Elsevier.
Mager, M. (2012). Data Analysis in Biochemistry and Biophysics. Burlington, Elsevier Science.
Wild, D. (2013). The immunoassay handbook: theory and applications of ligand binding, ELISA and related techniques. Elsevier Science.
