Enzyme Linked Immunosorbent Assay

Enzyme Linked Immunosorbent AssayIMMUNOLOGY PRATICAL grantENZYME LINKED IMMUNOSORBENT ASSAYmODULE NAME Clinical immuno recordyMODULE NUMBER APS6004MODULE LEADER DR JULIA REY-NORES assimilator NUMBER cc31761BSc (H) BMS 32014/2015IntroductionThe history of immunoassay was developed by Roalyn Yalow and Solomon Berson in 1950 rehearsed 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 chassis up by replacing the radio-isotopes with enzymes to make colouring material generation that was in 1960. 1, 2, 3More than 40 years, immunoassays use in different places, like testing ground medicine, hospitals and research to improve the health also for many an(prenominal) 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 det ect contaminants in food and water. Also, used as smell control to observe specific molecules used through product processing. 1Nowadays, the immunochemistry technology develops assays to try give as many dilution, mixing and measuring. Immunoassays atomic number 18 technique used to detect specific molecule. Its swear on the ingrained ability of antibody that be bind to the specific structure of molecules. This techniques are quick and right its depend on the antibody and antigen that found in the blood and tissue fluids. 1There are many type of immunoassays such(prenominal) as radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), and enzyme-linked immunoassay (enzyme-linked-immunosorbent serologic assay). 3,5In this report consign I will focus on enzyme-linked-immunosorbent serologic assay this technique that I used in the laboratory during 2 weeks to detect the antibody and the antigen. There is different types of ELISADirect ELISAIndirect ELIS A machinate ELISACompetitive ELISA 3Direct ELISAThis method technique depend on the antigen that surface in the surface of casing and the antibody of the patient and conjugated enzyme. 5 picture 1 shows move of direct ELISAThe indirect ELISAThe technique used the micro home plate cover with antigen. The primary antibody added to react with the antigen that situated 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 recrudesce the reaction colour that fixd the amount of the antibody. 3,7Figure 2 show the steps of the indirect ELISASandwich ELISASandwich ELISA is the technique that used to detect the antibody or antigen that are inclose in the patient blood. This technique also c completelyed capture method because it detect level of antigen amongst two layers of antibodies. The antigen to be measure in the technique should contain at le ast two antigenic epitope able-bodied of binding antibody. Sandwich ELISA has many advantages for example juicy 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 antibody antibody mouse anti-rabbit immunoglobulin G and polyclonal prat anti-rabbit IgG antibodies.To determine the dumbness of unidentified experiment X and Y.MaterialsCoating pilot light phosphate buffer saline (phosphate buffer solution) weaken 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 plate adaptable micropipettePractical week 1MethodsThis ste p was done by the lab technician to make the 96-well plate coated with antigen industrious to the students because its take long time.Figure 4 show the rabbit IgG antigen consecutive dilution by using degree Celsiusl coating bufferMonoclonal and polyclonal antibody procedure in tables 1 and 2Procedure of Monoclonal antibodyProcedure of Polyclonal antibodyAdded nose reardyl of diluents of buffer PBS from pillar 2-6 in the showtime plateAdded atomic number 6l 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 proceeding.Covered the plate and incubated at room temperature for 30 minutes.Washed 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 washing 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 come upThe colour will change to yellowThe result was read by spectrophotometerTable 2Figure 7 shows stepsWeek one result graph 1 shows the results of mouse anti-rabbit IgG monoclonal antibody titration in different dilutionResultGraph 2 shows results of goat anti-rabbit IgG HRP labelled antibody in different dilutionPractical week 2MethodStep done by lab te chnician coated 20 swell overnight with 100l/well of the capture antibody (monoclonal mouse anti-rabbit IgG ) and kept ready for use.( sandwich ELISA)Added 200l of rabbit 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 interpreted 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 freshAdded 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 washing 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 unti l colour developsAdded 50l stop reaction with (1 M HCL acid)Read absorbance at 450 nm by spectrophotometerThe resultGraph 3 shows standard calibration frizzle of rabbit 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 graph 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(p) dilution is 14000 because it gradually increase with slight antibodies and this dilution can detect the lowest concentration of antigen and also can be used for more poem 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 ((12000, 14000, 18000, 116000, 132000, 164000) the first dilution 12000 increase sharp until concentration of universal gravitational constant, 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 optimum 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 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 instant 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(prenominal) 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) concent ration 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. Clinical Chemistry, 52(7), pp.1430a-1431.2Tulsidas G, S. (2002). register 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 easy 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 Avai lable 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.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. 5961.32021.168810001.74111.46431.32791.46460.9640.61565001.34341.21980.5020.92950.74760.52772500.98110.86190.75280.58770.44640.22821250.6460.61520.60720.60480.49750.5081620.43330.40120.3850.3240.33320.3081310.25380.25880.24140.27370.2270.17250000000Table 2 the results of the absorbance of monoclonal antibody after subscription of the absorbance from blankconcentration1/20001/40001/80001/160001/320001/6400020003.15231.78830.91020.44660.19670.074910003.36381.54010.78440.35550.20670.10245002.79021.25640.58390.27160.12630.05232502.11931.00840.49330.23940.11540.04241251.51030.70360.64860.29590.14230.0539620.89260.5190.27590.12270.05450.1017310.59040.28110.14370.06140.0288-0.00040000000Table 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 sample de nsity 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 get 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