Clinical Immunology - ELISA Technique - Lab Report Example

Introduction: Enzyme-linked Immunoasborbent assay (Elisa) is a common analytic biochemistry assay involved in the determination of known analytes. Before its discovery, in 1960s, radioimmunoassay was the only technique in use for conducting immunoassays (Yalow & Berson, 1960) (Wide & Porath, 1966). As the name, suggest radioimmunoassay involved labelling of antigens or antibodies with radioactive material. The technique even though successful posed a potential health threat and was received with a lot of criticism. Consequently a safer alternative that would substitute the radioactive material with a nonradioactive signal. In the search for this alternative, two research groups independently and simultaneously described the use of enzymes as a replacement for radioisotopes. The first group was led by Peter Perlmann at Stockholm University Sweden and other group by Anton Schuurs at the Research Laboratories of NV Organin, Netherlands. The work by Perlman and Schuurs was built on the work by other researchers such as Villejuif who reported successful results of coupling antigens or antibodies with enzymes such as alkaline phosphatase in 1969 (Miles & Hales, 1968). Others include works by Avrameas who documented optimal coupling of the enzymes by use of glutaraldehyde. In 1971, Perlmann and Engvall published on the quantitative measurement of serum IgG of a rabbit using ELISA technique and alkaline phosphatase as the reporter label (Engvall & Perlman, 1971). The same year Van Weemen and Schuurs published on the quantification of human chorionic gonadotropin using horseradish peroxidise as the reporter (Miles & Hales, 1968). . Over the years following its discovery, the technique has evolved into a series of test formats such as direct and several “sandwich” variants. Today, ELISA is a routine technique that has a wide application in medical laboratories, manufactures of diagnostic products, pharmaceutical industries, veterinary medicine, and regulatory bodies among others (Lequin, 2005). This broad application of the technique has also resulted in the invention of different versions of the techniques. The general principle of ELISA is that an antigen or antibody is immobilised on a plate and the test antigen or antibody is added. The two react specifically, and excess test antigen or antibody is removed and the bound test substance is then detected using an enzymatic reaction. The technique can be run in quantitative and qualitative format depending on the aims of the experiment (Seppala, Rutanen, Heikinheimo, Jalanko, & Engvall 1978). In qualitative, the presence of colour may infer a positive reaction, while in quantitative assays the intensity of colour is proportional to the concentration of the analyte. There are two types of antibodies that are commonly used in ELISA assay. The first type is polyclonal antibodies; these are produced through activation of multiple B-lymphocytes the cells responsible for producing antibodies. Polyclonal antibodies therefore, have broad antigen specificity and different epitope affinities. They are normally of a high titre and affinity with robust detection due to multiple epitopes. The other types of antibodies common with ELISA technique are monoclonal antibodies. In contrast to polyclonal, monoclonal antibodies are produce by a single B-lymphocyte cultured in vitro. Monoclonal antibodies can be raised against any antigen and their specificity is known. Monoclonal antibodies have a lower background noise and cross reactivity due to their high specificity compared to polyclonal antibodies. This implies that test results produced by monoclonal antibodies are reproducible and more reliable than those produced by polyclonal antibodies are. These antibodies can be applied in about four types of ELISAs, which include; Direct, Indirect, competitive and sandwich ELISA. In direct ELISA, an antigen is directly attached to the solid phase of a micro-titre plate. During the assay a sample thought to contain antibodies specific to the coated antigen is added. These antibodies are normally labelled with an enzyme and upon addition of the substrate, a change in colour in the reaction mixture is observed. This technique however, is not efficient in measuring crude samples due cross-reactivity caused by contaminating proteins. These proteins also compete with antigen for binding sites in the plate. In indirect ELISA, antigens are coated on the plate just like in the direct ELISA. Primary test antibodies (sample­) are then added and antibodies specific to the coated antigens remain bound as excess antibodies are washed out. The bound antibodies are then detected using secondary antibodies specific to the bound antibodies. The secondary antibodies are tagged with an enzyme that reacts with the substrate to form visible colour. This technique is readily applied in cases of detecting reactive antibodies present in a given sample. Sandwich is another type of ELISA that is used to detect antigens of interest in a sample. Sometime sandwich ELISAs are also referred to as indirect. In this test format a known concentration of antibody, instead of the antigen of interest is coated in the microtitre plate. The coated antibody is referred to as capture antibody (Seppala, Rutanen, Heikinheimo, Jalanko, & Engvall, 1978). Any remaining binding sites on the plate are further blocked using non-reactive proteins such as bovine serum albumin. When the sample is added on the plate, antigens specific to capture antibodies are bound and remain during washing. Another specific antibody is the added that binds on the already bound antigens forming a sandwich. Enzyme tagged secondary antibodies against the primary antibody are added to the reaction mixture. These secondary antibodies bind specifically to the Fc portion of the primary antibodies. Any unbound antibodies are removed via washing and a substrate is added it is converted into a colour by the enzymes (Seppala, Rutanen, Heikinheimo, Jalanko, & Engvall, 1978). In some cases, several levels of sandwich are formed depending on the availability of enzyme linked reporter labels. ELISA can also be applied using competitive binding technique. In this type of ELISA, a non-labelled known concentration of antibody is incubated with its antigen present in a sample. The reaction mixture is then added in an antigen-coated plate, any unbound antibodies and the antibody/antigen complexes are removed during washing. This implies that the more antigens are present in the sample the more complexes are formed leaving only a few free antibodies that can bind to the antigen on the plate. A secondary antibody specific to the primary antibody and which is labelled with an enzyme is added on the plate. When a substrate is added a chromogenic signal is produced (Engvall, 1977). Sometimes in this type ELISA, the enzyme is coupled on the antigen rather than the antibody, this coupled antigen that competes for antibody binding sites with the test antigen. Aims: The aim of this experiment is to understand the significance of ELISA assay for investigation of disease in biomedical science. This practical was done in two sessions each with a different goal. The objective of the first part is to find out the optimum dilution for the monoclonal mouse anti-rabbit IgG antibodies and polyclonal goat anti-rabbit IgG antibodies by using different dilutions. The second objective done as the second part aimed at finding out the concentration of two unknown samples (X & Y) by drawing a standard curve using known concentrations of Rabbit IgG antibodies. Method: The following reagents were used in setting up this experiment, Coating buffer: phosphate buffer saline (PBS), Wash buffer: 0.05% Tween 20® in PBS, pH-7.4, Diluent: PBS, Blocking solution: 1 % (v/v) bovine serum albumin (BSA) in PBS, Antigen: rabbit IgG, Coating antibody: mouse monoclonal anti-rabbit IgG (dilution to be used determined on weeks 1 and 2), Detection antibody: Goat anti-rabbit IgG- Peroxidase conjugated (dilution to be used determined on weeks 1 and 2), Colour reagent (3,5,3’,5’tetramethylbenzidine (TMB; SureBlue, KPL), Stop solution (ClH 1M) Plates were already prepared as following: Firstly, 200ul of Rabbit IgG antigen was added to the first row (A1-A12) of the microtiter plate. As illustrated in yellow colour in the picture below. After that, 100ul of coating bufferwas added to the rest of the rows. As described in light blue in the figure below. Next step is transferring 100ul of rabbit IgG antigen from A to G well by doing serial dilution as can be seen in the figure below. Finally, the100ul rabbit IgG antigen was discarded after column (G) to keep column (H) as a BLANK. Different amount of rabbit IgG were incubated overnight, andin the following morning micotiter plates were washed 3 times with washing buffer, blocked for one hour with blocking buffer and washed again. Plates was kept in the fridge so plates are ready for the practical. Practical 1 (weeks 1 & 2): Monoclonal antibody (left half of the plate) The left half of the plate was used for the monoclonal antibodies. 200ul of monoclonal mouse anti-rabbit IgG was added to the first column as illustrated in the picture below with a blue colour. Colum 2 to 6 was filled with 100ul of diluents buffer PBS for the monoclonal antibodies as shown below. After that, 100ul of mouse anti-rabbit was transferred from A1 to A6 to dilute the sample and the 100ul of mouse anti rabbit IgG antibody was thrown away from well A6. The process was repeated for the other rows. Next step is incubating the plate for 30 minutes at room temperature. The plate was washed three times by Phosphate Buffer Saline (only for the left half). It is important to cover the other half of the plate with sticker. Polyclonal antibody titration (right half) The cover of the polyclonal part was taken off and 100ul of diluents buffer PBS was added to columns 8 to 12. And 200ul of goat anti rabbit IgG-HRP was added to column number 7. After that, polyclonal Ab dilutions were prepared as discussed in monoclonal step. 100ul was goat anti-mouse IgG-HRP(fixed dilution 1/5000) was added to all wells in the left half as illustrated bellow: Next, the plate was incubated at room temperature for 30 minutes. The plate was washed three times with washing buffer (make sure wells have no bubbles). The plate was protected from the light until colour develop The reaction was stopped by adding 50 ml of ClH 1 M to the wells. Finally, microtiter plate can be read by using spectrophotometer at (450 nm), and now graph can be drawn. Practical 2 (week 15 & 16) Method: Plate has been coated with mouse anti-rabbit IgG monoclonal antibody. After incubation overnight, it was washed, blocked and washed again. 100ul of PBS was added to columns 1 and 2. 100ul of unknown sample (X) was added to A3 and A4. 100ul of unknown sample (Y) was added to columns B3 and B4. A 1 and A2 was filled with 200ul of rabbit IgG (2ug/ul). Serial dilutions wasdone by transferring 100ul from the first well in column 1 (A1) to (G1) and throw away the mixture after G1. The same was done for the second column. The plate was incubated at room temperature for 30 minutes while it was covered by sticker. The plate was washed with (PBS/Tween). 100ul of goat anti-rabbit IgG-HRP was added to the all wells which have samples. Then, the plate was Incubated for 30 minutes and washed three times with (PSB/Tween ) TMB substrate (100ul/well) was added and then the plate was hidden from the light until the colour developed. Finally, the reaction was stopped by1 M HCL (50 µl/well) and absorbance has been read at 450nm. Result: Week1: Table1: Shows the absorbance of Mouse Anti-Rabbit IgG in different dilutions before decreasing the blank value from each sample. Table 1: Showing the OD values of Anti-Rabbit IgG. 1 2 3 4 5 6 2000 2.6189 2.77 2.7905 2.4752 1.1247 0.5197 1000 2.3413 2.2714 2.2627 2.1128 1.7101 1.3508 500 1.8122 1.6982 1.6613 1.6172 1.2577 0.9391 250 1.2901 1.2872 1.2437 1.2061 1.0529 0.8659 125 0.8905 0.897 0.8485 0.7773 0.6954 0.5498 62 0.5342 0.506 0.4864 0.4544 0.3886 0.2696 31 0.3492 0.3215 0.3362 0.2955 0.1965 0.132 0 0.2473 0.1393 0.1464 0.107 0.0659 0.1317 Table2: Shows the absorbance of Mouse Anti-Rabbit IgG in different dilutions after decreasing the blank value from each sample. Table 2 2000 2.3716 2.6307 2.6441 2.3682 1.0588 0.388 1000 2.094 2.1321 2.1163 2.0058 1.6442 1.2191 500 1.5649 1.5589 1.5149 1.5102 1.1918 0.8074 250 1.0428 1.1479 1.0973 1.0991 0.987 0.7342 125 0.6432 0.7577 0.7021 0.6703 0.6295 0.4181 62 0.2869 0.3667 0.34 0.3474 0.3227 0.1379 31 0.1019 0.1822 0.1898 0.1885 0.1306 0.0003 0 0 0 0 0 0 0 Graph 1: The standard curve achieved from table 2. This is used to determine the best dilution of the Mouse anti-Rabbit IgG monoclonal antibody. Table 3: The table below shows the absorbance reading of Goat Anti-RabitIgG HRP-conjugated polyclonal antibody before decreasing the blank value from each sample. Absorbance Antigen concentration ng/ml 1:2000 1:4000 1:8000 1:16000 1:32000 1:64000 7 8 9 10 11 12 2000 3.093 3.6147 1.969 0.8483 0.5309 0.2867 1000 3.2067 3.059 1.8123 0.9135 0.5282 0.2919 500 3.4298 2.681 1.3623 0.6703 0.3615 0.2105 250 3.3215 2.2193 1.1872 0.6455 0.3119 0.1296 125 2.7127 1.6158 0.8915 0.4545 0.2372 0.1433 62 1.7718 0.9757 0.5679 0.2502 0.1225 0.0791 31 1.1735 0.6316 0.1593 0.116 0.0728 0.0461 0 0.0863 0.0489 0.0382 0.0364 0.0297 0.0225 Table 4: The table below shows the absorbance reading of Goat Anti-RabitIgGhrp-conjugated polyclonal antibody after decreasing the blank value from each sample absorbance Antigen concentration ng/ml 1:2000 1:4000 1:8000 1:16000 1:32000 1:64000 2000 3.0067 3.5658 1.9308 0.8119 0.5012 0.2642 1000 3.1204 3.0101 1.7741 0.8771 0.4985 0.2694 500 3.3435 2.6321 1.3241 0.6339 0.3318 0.188 250 3.2352 2.1704 1.149 0.6091 0.2822 0.1071 125 2.6264 1.5669 0.8533 0.4181 0.2075 0.1208 62 1.6855 0.9268 0.5297 0.2138 0.0928 0.0566 31 1.0872 0.5827 0.1211 0.0796 0.0431 0.0236 0 0 0 0 0 0 0 Graph 2: The graph belowshows the standard curves of the absorbance readings that are displayed in table 4. Table 5& 6: The table below shows the standard Rabbit IgG in different concentration with their average absorbance readings after decreasing the blank value from each well. Duplicate absorbance of standard 0.5435 0.4234 0.4708 0.3926 0.421 0.3596 0.3446 0.3247 0.2806 0.2609 0.2457 0.2031 0.1649 0.1504 0.1154 0.0988 Concentration dilution Average of duplicate absorbance 2000 0.37635 1000 0.3246 500 0.2832 250 0.22755 125 0.16365 62 0.1173 31 0.05055 0 0 Table 7: the average absorbance of (X) and (Y) after decreasing the blank value from each sample. X 0.2766 Y 0.12735 Blank 0 Graph 3: The graph below shows the calibration curve of the antigen (Rabbit IgG) to determine the concentrations of X & Y. Graph 4: The graph below shows the logarithmic curve of the antigen (Rabbit IgG). Y = 0.0777ln(x) - 0.2085 Y = SAMPLE ABSORBANCE FOR X AND Y X= SAMPLE CONCENTRATION 0.2766 = 0.0777 (X) – 0.2085 0.2766 + 0.2085 = 0.4851 (X) 0.4851 / 0.0777 = X HENCE (X) = 6.243 SO THE INTI LOG WILL BE = 514.39 ng Y= sample concentration 0.12735= 0.0777 (x) – 0.2085 0.12735+ 0.2085 = 0.0777 (x) 0.3358 / 0.0777 = x So X = 4.322 The anti-log of 4.322 will be = 75.339 ng Discussion: ELISA is one of the test techniques that can be easily customized and optimized to achieve high throughput assays. The test utilizes determination of colour intensity as an indirect correlate to concentration of the analyte under investigation. Among the optimatisation steps is in indentifying the optimal dilution that gives the best signal that can give a true estimate of the concentration. This set involves serially diluting the reagents and obtaining an OD value of each dilution. The dilution giving the highest OD is chosen as an optimal concentration for the setup. According to the results of this experiment, the optimal concentration for monoclonal mouse anti-rabbit IgG is between 1:400 and 1:800. In contrast, the optimal concentration for the polyclonal goat anti-rabbit IgG is between 1:1600 and 1:3200. This implies that monoclonal antibodies are of low titre hence the higher concentration (low dilution) required to achieve optimal signal (Engvall, 1977). It may also imply that monoclonal antibodies have a high specificity therefore can only reacted with a limited number of the antigens present in the sample hence the high concentration needed. On the other hand, polyclonal antibodies are of high titre and have broad specificity, cross which they can interact with antigens. The implication of this is that polyclonal antibodies can give a strong signal even in weak concentrations (Ljungstrom, Engvall & Ruitenberg, 1974). The second part of this experiment was to quantitatively determine the concentration of two samples X and Y using a known standard of anti-rabbit IgG. The known standard was used to plot a calibration curve upon which the concentration of the unknowns was determined. The standard gave an expected curve shown in figure 3 above. The test samples X and Y were done in duplicates to minimize inter-reading variation. According the calibration curve the concentration of X and Y was determined as 514.39 ng and 75.339 ng respectively. It is important to note that these two concentration were within the expected range and therefore acceptable. There was a slight variation between the duplicates of both the standard and the test samples. Such variation may have been caused by inherent sources such as minor errors in pipetting, differences in incubation time and mixing. There were no cases of contamination noted in both experiments. ELISA techniques are prone to contamination and some antigens can be lost during the washing steps (Engvall, 1977). It is therefore critical to handle the experiment carefully in order to produce reliable results. Duplicating the test setup is also handy in noting variation caused by extrinsic factors such as pipetting errors or cross contamination of wells. Conclusion: In conclusion, ELISA is a scientific technique useful in the qualitative and quantitative investigation samples. It is a technique that was invented by two groups of independent researchers and has highly evolved. In this experiment, ELISA was used to determine the optimal concentration of monoclonal and polyclonal mouse and goat anti-rabbit IgG. According to this test, monoclonal antibodies are of low titre and require higher concentration than polyclonal antibodies. After optimization, the technique was used to determine that concentration of two unknown samples X and Y. References Engvall E, Perlman P. 1971. "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 871–4. Engvall E. 1977. ‘Quantitative enzyme immunoassay (ELISA) in microbiology.’ Med Biol 55:193–200. Miles LEM, Hales CN. 1968. ‘Labelled antibodies and immunological assay systems.’ Nature 219:186 –9. Lequin R 2005. "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)". Clin. Chem. 51 (12): 2415–8. Ljungstrom I, Engvall E, Ruitenberg EJ. Proceedings: 1974. ‘ELISA, enzyme-linked immunosorbent assay a new technique for serodiagnosis of trichinosis.’ Parasitology 69:xxiv. Seppala M, Rutanen EM, Heikinheimo M, Jalanko H, Engvall E. 1978. ‘Detection of trophoblastic tumour activity by pregnancy-specific 1 glycoprotein.’ Int J Cancer;21:265–7. Yalow RS, Berson SA. 1960. ‘Immunoassay of endogenous plasma insulin in man.’ Clin Invest;39:1157–75. Wide L, Porath J. 1966. ‘Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies.’ Biochem Biophys Acta 30:257–260. Read More