Enzyme Linked Immunosorbent Assay

Enzyme Linked Immunosorbent hindranceIMMUNOLOGY PRATICAL ASSIGNMENTENZYME LINKED IMMUNOSORBENT ASSAYmODULE NAME clinical immuno put downyMODULE NUMBER APS6004MODULE LEADER DR JULIA REY-NORESSTUDENT NUMBER cc31761BSc (H) BMS 32014/2015IntroductionThe 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 discover 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 illusion generation that was in 1960. 1, 2, 3More than 40 years, immunoassays use in different places, like laboratory medicine, hospitals and research to improve the health excessively for numerous 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 i n food and water. Also, used as quality control to observe specific molecules used through product processing. 1Nowadays, the immunochemistry technology develops assays to try obviate as many dilution, mixing and measuring. Immunoassays atomic number 18 technique used to detect specific molecule. Its rely on the ingrained ability of antibody that are arrest to the specific structure of molecules. This techniques are quick and accurate its depend on the antibody and antigen that found in the blood and tissue fluids. 1There are many type of immunoassays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), and enzyme-linked immunoassay (enzyme-linked-immunosorbent serologic assay). 3,5In 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 ELISADirect ELISAIndirect ELISA organise ELISACompetitive ELISA 3Direct ELISAThis method techni que depend on the antigen that coated in the surface of home plate and the antibody of the patient and immix enzyme. 5 contrive 1 shows move of direct ELISAThe indirect ELISAThe technique used the micro plate coated with antigen. The primary antibody added to react with the antigen that wintry 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 tinge that determined the amount of the antibody. 3,7 approach pattern 2 show the steps of the indirect ELISASandwich ELISASandwich ELISA is the technique that used to detect the antibody or antigen that are wassail 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 support at least two antigenic epitope capable of binding antibody. Sandwich ELISA h as many advantages for example high specificity, flexibility and sensitivity. 3,8Figure 3 shows the steps of sandwich ELISAThe aim of practicalTo achieve a grid experimental to detect the optimal detection and capture antibody titration, by using monoclonal antibody antibody mouse anti- dassie IgG and polyclonal fanny anti-rabbit IgG antibodies.To determine the concentration of un cognize sample X and Y.MaterialsCoating caramel phosphate buffer saline (PBS) airstream buffer 0.05% Tween 20in PBS, pH 7.4Diluent PBSBlocking solution 1% (w/v) bovine serum albumin (BSA) in PBSAntigen rabbit IgGCoating antibody Mouse anti-rabbit IgG monoclonal antibodyDetection antibody Goat anti-rabbit IgG peroxidase conjugatedColour reagent .tetramethyl benzidine (TMB)Stop solution (1M HCL)96-well micro plateadjustable micropipettePractical week 1MethodsThis step was done by the lab technician to make the 96-well plate coated with antigen coiffure to the students because its take long time.Figure 4 show the rabbit IgG antigen serial dilution by using hundredl coating bufferMonoclonal and polyclonal antibody procedure in tables 1 and 2Procedure of Monoclonal antibodyProcedure of Polyclonal antibodyAdded degree centigradel of diluents of buffer PBS from column 2-6 in the first plateAdded light speedl of buffer PBS from column 8-12 in the second half of the plateAdded 200l of monoclonal mouse anti-rabbit IgG from A1well to H1 wellAdded 200l of goat anti-rabbit IgG HRP to column 7transferred 100l by doing serial dilution mixed well from column 1 to columns 2 ,3,4,5,6 then discard 100l from well 6Transferred 100l by doing serial dilution from column 7 to columns 8,9,10,11,12 then 100l 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. rinse the plate three times with wash bufferWashed the plate three times with wash bufferTable 1The final steps100l of goat anti -mouse IgG-HRP was added to columns 1 to 6200l of goat anti-rabbit IgG-HRP was added to columns 7 to 12The plate was covered and incubated at room temperature for 30 minutesThe plate was washed three times with the lavation buffer100l 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 50l of stop reaction 1 M HCL was added to all wellsThe colour will change to yellowThe essence was read by spectrophotometerTable 2Figure 7 shows stepsWeek one resultGraph 1 shows the results of mouse anti-rabbit IgG monoclonal antibody titration in different dilution runGraph 2 shows results of goat anti-rabbit IgG HRP tagged antibody in different dilutionPractical week 2Method note done by lab technician coated 20 wells overnight with 100l/well of the capture antibody (monoclonal mouse anti-rabbit IgG ) and kept ready for use.( sandwich ELISA)Added 200l of r abbit IgG to well A1 and A2Added 100l of PBS diluents to wells from B to H in column 1and 2From A1, 100l rabbit IgG was taken and added to B1 then serial dilution take place up to G1 then 100l was discarded from G1100l of rabbit IgG was taken from A2 and added to B2 then continued the serial dilution up to G2 then 100l from G2 discardedWell H1 and H2 was used as fatuousAdded 100l of unknown sample X to wells (A3 and A4)Added 100l of unknown sample Y to wells (B3 and B4)Incubated the plate for 30 minutes at room temperatureWashed the plate 3 times with process buffer PBSAdded 100l of goat anti-rabbit IgG HRP labelled to all 20 wellsIncubated the plate for 30 minutes at room temperatureWashed the plate 3 times with Buffer BPSAdded 100l TMB substrate to all 20 wellsIncubated the plate and protected from the light until colour developsAdded 50l stop reaction with (1 M HCL acid) immortalize absorbance at 450 nm by spectrophotometerThe resultGraph 3 shows standard calibration thin of r abbit IgGGraph 4 shows the equation log of concentration rabbit IgGCalculation of samplesTable 3 shows the calculation to found the concentration of samples X and YDiscussionFrom the result that shows in interpret one there are six curves of the monoclonal mouse anti-rabbit IgG with different serious dilutions(12000, 14000, 18000, 116000, 132000, 164000). From my result, the dilution 12000 is increase fast and it consumption more antibodies which is not recommended. The best dilution is 14000 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 18000 it increase but is less than dilution 14000 it need more antibody, while the dilutions (116000, 132000, 164000) 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, 14000, 18000, 116000, 132000, 164000) the first dilution 12000 increase sharp until concentration of potassium, then decrease slowly up concentration of 2000 so this dilution not recommended due to over opsonisation of antibodies. The second, dilution 14000 increased gradually and it need less antibody and can detect the lowest concentration of antigen so it is the optimal for the goat anti-rabbit IgG HRP labelled antibody. Third dilution 18000 is increase slow and require more antibody. The last three dilutions, 116000, 132000, and 164000 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 importan t things such as the best technique to choose the dilution of antibody and antigen detection. Also, to discriminate between the best antibody to detect antigen.There are many factors that affect the result of ELISA like the incubation time should be 1 min 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.ConclusionIn 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.Reference1-Avrameas, S. (2006). Historical Background of the Invention of EIA and ELISA. Clini cal Chemistry, 52(7), pp.1430a-1431.2Tulsidas G, S. (2002). narration 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 for sale 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. 20146-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 A ccessed 27 Nov. 2014.AppendixResult week one practical1234567891011122.11042.12921.96131.66371.39741.25743.25281.84490.95610.49390.24650.13381.82081.54991.40531.53231.04120.70423.46431.59670.83030.40280.25650.16131.42311.30540.57940.99720.82480.61632.89071.3130.62980.31890.17610.11121.06080.94750.83020.65540.52360.31682.21981.0650.53920.28670.16520.10130.72570.70080.68460.67250.57470.59671.61080.76020.69450.34320.19210.11280.5130.48680.46240.39170.41040.39670.99310.57560.32180.170.10430.16060.33350.34440.31880.34140.30420.26110.69090.33770.18960.10870.07860.05850.07970.08560.07740.06770.07720.08860.10050.05660.04590.04730.04980.0589Table 1 shows the result of the absorbance of monoclonal antibody and polyclonal antibodyconcentration1/20001/40001/80001/160001/320001/6400020002.03072.04361.88391.5961.32021.168810001.74111.46431.32791.46460.9640.61565001.34341.21980.5020.92950.74760.52772500.98110.86190.75280.58770.44640.22821250.6460.61520.60720.60480.49750.5081620.43330.40120.3850.32 40.33320.3081310.25380.25880.24140.27370.2270.17250000000Table 2 the results of the absorbance of monoclonal antibody after subscription of the absorbance from vacuousconcentration1/20001/40001/80001/160001/320001/6400020003.15231.78830.91020.44660.19670.074910003.36381.54010.78440.35550.20670.10245002.79021.25640.58390.27160.12630.05232502.11931.00840.49330.23940.11540.04241251.51030.70360.64860.29590.14230.0539620.89260.5190.27590.12270.05450.1017310.59040.28110.14370.06140.0288-0.00040000000Table 3 shows the results of polyclonal antibody after subscription of blankResult of week 2 practical1234A0.60840.54260.43060.419B0.56990.45890.24250.2505C0.56020.4504D0.50850.4093E0.42380.3164F0.30040.2355G0.19970.1794H0.12420.1093Table 4 shows the result of rabbit IgG absorbance and two unknown sampleConcentration IgG (ng/ml)12meanmean- blanksamplesamplemeanmean- blank20000.60840.54260.57550.45875X=0.4306X=0.4190.42480.30810000.56990.45890.51440.39765Y=0.2425Y=0.25050.24650.12975000.56020. 45040.50530.388552500.50850.40930.45890.342151250.42380.31640.37010.25335620.30040.23550.267950.1512310.19970.17940.189550.072800.12420.10930.116750Table 5 shows the steps of rabbit IgG and two unknown sample, mean then subscription of blank to make calibration curve and equation to take in the concentration of sample X and XAbbreviationRIA RadioimmunoassayEIA Enzyme immunoassayFIA Fluorescent immunoassayELISA Enzyme-linked immunosorbent assayPBS Phosphate buffer salineBSA bovine serum albuminTMB Tetramethyl benzidineHRP Horseradish peroxidase1M HCL 1 molar of Hydrochloric acidLog LogarithmY AbsorbanceIgG immunoglobulin GX Concentration Result ignore