Enzyme Linked Immunosorbent Assay

Enzyme Linked Immunosorbent Assay IMMUNOLOGY PRATICAL ASSIGNMENT ENZYME LINKED IMMUNOSORBENT ASSAY mODULE NAME: Clinical immunology MODULE NUMBER: APS6004 MODULE LEADER: DR JULIA REY-NORES STUDENT NUMBER: 20031761 BSc (H) BMS 3 2014/2015 Introduction The history of immunoassay was developed by Roalyn Yalow and Solomon Berson in 1950 used the Radio-immunoassay (RIA) and they awarded in 1977 Nobel Prize because they developed RIA to detect and measure the level of glucose in the blood for diabetic patient. However, the technology was build up by replacing the radio-isotopes with enzymes to make colour generation that was in 1960. ]1, 2, 3[ More than 40 years, immunoassays use in different places, like laboratory medicine, hospitals and research to improve the health also for many purposes. In addition, immunoassay use in life science research to study the biology system by chase different, hormones, proteins and antibody. However, it use in industry to detect contaminants in food and water. Also, used as quality control to observe specific molecules used through product processing. ]1[ Nowadays, the immunochemistry technology develops assays to try eliminate as many dilution, mixing and measuring. Immunoassays are technique used to detect specific molecule. It’s rely on the ingrained ability of antibody that are bind to the specific structure of molecules. This techniques are quick and accurate it’s depend on the antibody and antigen that found in the blood and tissue fluids. ]1[ There are many type of immunoassays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), and enzyme-linked immunoassay (ELISA). ]3,5[ In this report file I will focus on ELISA this technique that I used in the laboratory during two weeks to detect the antibody and the antigen. There is different types of ELISA: Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA [3] Direct ELISA This method technique depend on the antigen that coated in the surface of plate and the antibody of the patient and conjugated enzyme. ]5[ Figure 1 shows steps of direct ELISA The indirect ELISA The technique used the micro plate coated with antigen. The primary antibody added to react with the antigen that fixed to the plate. Then washed away. Added enzyme conjugated secondary antibody anti-isotope antibody which binds to the primary antibody. After that washed away and added the substrate enzyme to produce the reaction colour that determined the amount of the antibody. ]3,7[ Figure 2 show the steps of the indirect ELISA Sandwich ELISA Sandwich ELISA is the technique that used to detect the antibody or antigen that are present in the patient blood. This technique also called capture method because it detect level of antigen between two layers of antibodies. The antigen to be measure in the technique should contain at least two antigenic epitope capable of binding antibody. Sandwich ELISA has many advantages for example high specificity, flexibility and sensitivity. ]3,8[ Figure 3 shows the steps of sandwich ELISA The aim of practical: To achieve a grid experimental to detect the optimal detection and capture antibody titration, by using monoclonal mouse anti-rabbit IgG and polyclonal goat anti-rabbit IgG antibodies. To determine the concentration of unknown sample X and Y. Materials: Coating buffer: phosphate buffer saline (PBS) Wash buffer: 0.05% Tween 20 ®in PBS, pH 7.4 Diluent: PBS Blocking solution: 1% (w/v) bovine serum albumin (BSA) in PBS Antigen : rabbit IgG Coating antibody: Mouse anti-rabbit IgG monoclonal antibody Detection antibody :Goat anti-rabbit IgG – peroxidase conjugated Colour reagent .tetramethyl benzidine (TMB) Stop solution (1M HCL) 96-well micro plate Adjustable micropipette Practical week 1 Methods: This step was done by the lab technician to make the 96-well plate coated with antigen ready to the students because it’s take long time. Figure 4 show the rabbit IgG antigen serial dilution by using 100 µl coating buffer Monoclonal and polyclonal antibody procedure in tables 1 and 2: Procedure of Monoclonal antibody Procedure of Polyclonal antibody Added 100 µl of diluents of buffer PBS from column 2-6 in the first plate Added 100 µl of buffer PBS from column 8-12 in the second half of the plate Added 200 µl of monoclonal mouse anti-rabbit IgG from A1well to H1 well Added 200 µl of goat anti-rabbit IgG HRP to column 7 transferred 100 µl by doing serial dilution mixed well from column 1 to columns 2 ,3,4,5,6 then discard 100 µl from well 6 Transferred 100 µl by doing serial dilution from column 7 to columns 8,9,10,11,12 then 100 µl discard from colum12 (mixed well) Covered the plate and incubated at room temperature for 30 minutes. Covered the plate and incubated at room temperature for 30 minutes. Washed the plate three times with wash buffer Washed the plate three times with wash buffer Table 1 The final steps 100 µl of goat anti-mouse IgG-HRP was added to columns 1 to 6 200 µl of goat anti-rabbit IgG-HRP was added to columns 7 to 12 The plate was covered and incubated at room temperature for 30 minutes The plate was washed three times with the washing buffer 100 µl of substrate (TMP) was added to all the columns from 1-12 and incubated at room temperature and the plate was observed to check the change of colour to blue colour. After the colour become blue 50 µl of stop reaction 1 M HCL was added to all wells The colour will change to yellow The result was read by spectrophotometer Table 2 Figure 7 shows steps Week one: result Graph 1 shows the results of mouse anti-rabbit IgG monoclonal antibody titration in different dilution Result Graph 2 shows results of goat anti-rabbit IgG HRP labelled antibody in different dilution Practical week 2 Method: Step done by lab technician coated 20 wells overnight with 100 µl/well of the capture antibody (monoclonal mouse anti-rabbit IgG ) and kept ready for use.( sandwich ELISA) Added 200 µl of rabbit IgG to well A1 and A2 Added 100 µl of PBS diluents to wells from B to H in column 1and 2 From A1, 100 µl rabbit IgG was taken and added to B1 then serial dilution take place up to G1 then 100 µl was discarded from G1 100 µl of rabbit IgG was taken from A2 and added to B2 then continued the serial dilution up to G2 then 100 µl from G2 discarded Well H1 and H2 was used as blank Added 100 µl of unknown sample X to wells (A3 and A4) Added 100 µl of unknown sample Y to wells (B3 and B4) Incubated the plate for 30 minutes at room temperature Washed the plate 3 times with washing buffer PBS Added 100 µl of goat anti-rabbit IgG HRP labelled to all 20 wells Incubated the plate for 30 minutes at room temperature Washed the plate 3 times with Buffer BPS Added 100 µl TMB substrate to all 20 wells Incubated the plate and protected from the light until colour develops Added 50 µl stop reaction with (1 M HCL acid) Read absorbance at 450 nm by spectrophotometer The result: Graph 3 shows standard calibration curve of rabbit IgG Graph 4 shows the equation log of concentration rabbit IgG Calculation of samples Table 3 shows the calculation to found the concentration of samples X and Y Discussion From the result that shows in graph one there are six curves of the monoclonal mouse anti-rabbit IgG with different serious dilutions(1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000). From my result, the dilution 1:2000 is increase fast and it consumption more antibodies which is not recommended. The best dilution is 1:4000 because it gradually increase with less antibodies and this dilution can detect the lowest concentration of antigen and also can be used for more numbers of samples. However, the dilution 1:8000 it increase but is less than dilution 1:4000 it need more antibody, while the dilutions (1:16000, 1:32000, 1:64000) need more antibodies and not detect antigen in low concentration. The graph 2 shows the result of polyclonal antibody and the graph has sex different curves with different serious dilutions ((1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000) the first dilution 1:2000 increase sharp until concentration of 1000, then decrease slowly up concentration of 2000 so this dilution not recommended due to over opsonisation of antibodies. The second, dilution 1:4000 increased gradually and it need less antibody and can detect the lowest concentration of antigen so it is the optimum for the goat anti-rabbit IgG HRP labelled antibody. Third dilution 1:8000 is increase slow and require more antibody. The last three dilutions, 1:16000, 1:32000, and 1:64000 are not showing significant elevation when increase the concentration and cannot used because it not detect high absorbance of antigen. The graph 3 shows the calibration curve of the known concentration to determine the concentration of two unknown samples X and Y. the graph 4 shows the equation make by log concentration of calibration curve to calculated the concentration of unknown samples. During this practical I learned a lot of important things such as the best technique to choose the dilution of antibody and antigen detection. Also, to compare between the best antibody to detect antigen. There are many factors that affect the result of ELISA like the incubation time should be 1 hour but we reduced to 30 minutes and this not enough for the reaction take place between antibody and antigen, manual washing cause insufficient washing and mixed with other micro plate wells. The pipettes some time not working due to some problem of tips. Conclusion In conclusion, the optimum monoclonal Mouse anti-rabbit IgG antibody concentration is 1/4000, while the optimum polyclonal Goat anti-rabbit IgG HRP labelled antibody concentration is 1/4000, and the concentration of unknown sample( X )is 287ng/ml and unknown of sample (Y) concentration is 41ng/ml. the ELISA is the best technique to detect the reaction between antibody and antigen. Reference 1-Avrameas, S. (2006). Historical Background of the Invention of EIA and ELISA. Clinical Chemistry, 52(7), pp.1430a-1431. 2Tulsidas G, S. (2002). HISTORY AND DEVELOPMENT OF ANALYTICAL CHEMISTRY IN LIFE SCIENCES WITH REFERENCE TO IMMUNOASSAY IN MEDICINE. Health and Population, 3(25), pp.140-147. 3- Owen, J. et al. 2009. Immunology by Kuby. 7th ed. New York: W. H Freeman and Company. 4-Immunochemistry.com, (2014). Apoptosis, Caspases, Assay Development, ELISA Buffers, ELISA Detection. [online] Available at: http://www.immunochemistry.com [Accessed 26 Nov. 2014]. 5-Accelero-bioanalytics.com, (2014). Home Accelero ® Bioanalytics GmbH. [online] Available at: http://www.accelero-bioanalytics.com [Accessed 27 Nov. 2014] 6-Wieslab.com, (2014). Wieslab Laboratory Services Home. [online] Available at: http://www.wieslab.com [Accessed 27 Nov. 2014]. 7-Pharmatutor.org, (2014). Articles | PharmaTutor. [online] Available at: http://www.pharmatutor.org/articles [Accessed 27 Nov. 2014]. 8-Elisa-antibody.com, (2014). ELISA Antibody, protocol and troubleshooting. [online] Available at: http://www.elisa-antibody.com [Accessed 27 Nov. 2014]. Appendix Result week one practical 1 2 3 4 5 6 7 8 9 10 11 12 2.1104 2.1292 1.9613 1.6637 1.3974 1.2574 3.2528 1.8449 0.9561 0.4939 0.2465 0.1338 1.8208 1.5499 1.4053 1.5323 1.0412 0.7042 3.4643 1.5967 0.8303 0.4028 0.2565 0.1613 1.4231 1.3054 0.5794 0.9972 0.8248 0.6163 2.8907 1.313 0.6298 0.3189 0.1761 0.1112 1.0608 0.9475 0.8302 0.6554 0.5236 0.3168 2.2198 1.065 0.5392 0.2867 0.1652 0.1013 0.7257 0.7008 0.6846 0.6725 0.5747 0.5967 1.6108 0.7602 0.6945 0.3432 0.1921 0.1128 0.513 0.4868 0.4624 0.3917 0.4104 0.3967 0.9931 0.5756 0.3218 0.17 0.1043 0.1606 0.3335 0.3444 0.3188 0.3414 0.3042 0.2611 0.6909 0.3377 0.1896 0.1087 0.0786 0.0585 0.0797 0.0856 0.0774 0.0677 0.0772 0.0886 0.1005 0.0566 0.0459 0.0473 0.0498 0.0589 Table 1 shows the result of the absorbance of monoclonal antibody and polyclonal antibody concentration 1/2000 1/4000 1/8000 1/16000 1/32000 1/64000 2000 2.0307 2.0436 1.8839 1.596 1.3202 1.1688 1000 1.7411 1.4643 1.3279 1.4646 0.964 0.6156 500 1.3434 1.2198 0.502 0.9295 0.7476 0.5277 250 0.9811 0.8619 0.7528 0.5877 0.4464 0.2282 125 0.646 0.6152 0.6072 0.6048 0.4975 0.5081 62 0.4333 0.4012 0.385 0.324 0.3332 0.3081 31 0.2538 0.2588 0.2414 0.2737 0.227 0.1725 0 0 0 0 0 0 0 Table 2 the results of the absorbance of monoclonal antibody after subscription of the absorbance from blank concentration 1/2000 1/4000 1/8000 1/16000 1/32000 1/64000 2000 3.1523 1.7883 0.9102 0.4466 0.1967 0.0749 1000 3.3638 1.5401 0.7844 0.3555 0.2067 0.1024 500 2.7902 1.2564 0.5839 0.2716 0.1263 0.0523 250 2.1193 1.0084 0.4933 0.2394 0.1154 0.0424 125 1.5103 0.7036 0.6486 0.2959 0.1423 0.0539 62 0.8926 0.519 0.2759 0.1227 0.0545 0.1017 31 0.5904 0.2811 0.1437 0.0614 0.0288 -0.0004 0 0 0 0 0 0 0 Table 3 shows the results of polyclonal antibody after subscription of blank Result of week 2 practical 1 2 3 4 A 0.6084 0.5426 0.4306 0.419 B 0.5699 0.4589 0.2425 0.2505 C 0.5602 0.4504 D 0.5085 0.4093 E 0.4238 0.3164 F 0.3004 0.2355 G 0.1997 0.1794 H 0.1242 0.1093 Table 4 shows the result of rabbit IgG absorbance and two unknown sample Concentration IgG (ng/ml) 1 2 mean mean- blank sample sample mean mean- blank 2000 0.6084 0.5426 0.5755 0.45875 X=0.4306 X=0.419 0.4248 0.308 1000 0.5699 0.4589 0.5144 0.39765 Y=0.2425 Y=0.2505 0.2465 0.1297 500 0.5602 0.4504 0.5053 0.38855 250 0.5085 0.4093 0.4589 0.34215 125 0.4238 0.3164 0.3701 0.25335 62 0.3004 0.2355 0.26795 0.1512 31 0.1997 0.1794 0.18955 0.0728 0 0.1242 0.1093 0.11675 0 Table 5 shows the steps of rabbit IgG and two unknown sample, mean then subscription of blank to make calibration curve and equation to get the concentration of sample X and X Abbreviation RIA Radioimmunoassay EIA Enzyme immunoassay FIA Fluorescent immunoassay ELISA Enzyme-linked immunosorbent assay PBS Phosphate buffer saline BSA Bovine serum albumin TMB Tetramethyl benzidine HRP Horseradish peroxidase 1M HCL 1 molar of Hydrochloric acid Log Logarithm Y Absorbance IgG immunoglobulin G X Concentration Result ignore