Ouchterlony Double Diffusion Assay Biology Essay

Ouchterlony Double dispersal examine Biology EssayIntroductionPolyclonal antibodies be produced by different B- lymphocytes in response to the same antigen, which recognise different parts of the antigen. Because the human resistant system cannot know in advance what pathogens it result confront, it prepares for future infections by creating millions of different antibodies. Each of these highly selective proteins recognizes and binds to a specific target, or antigen, then signals other comp one(a)nts of the immune system to destroy the target. These naturally-occurring polyclonal antibodies play a crucial role in triggering an immune responsePolyclonal antibodies are routinely used as ligands for the preparation of immunoaffinity columns labeling reagents for the qualitative and quantitative determination of molecules in a variety of assays, such as double airing, radial immuno-diffusion, ammonium sulfate precipitation and ion diversify chromatography.AimThe aim of this practi cal is to differentiate the civilization of serum immunoglobulin G by ammonium sulphate and ion swop chromatography.The first katharsis step entrust normally involve a effectuate such as divisional precipitation with increasing concentrations of ammonium sulphate. This method is not designed to achieve total purgation, only when to contract as much contaminant protein as possible whilst retaining all the protein of interest. virtually proteins entrust precipitate from firmness of purpose at high table salt concentrations, but the salt concentration required to precipitate them varies considerably. Ammonium sulphate get out be used as it is possible to set up salt concentration which will differentially precipitate serum proteins. Ammonium sulphate precipitation procedure was carried out to disjoined the serum proteins into four reckons. A calculate containing the serum protein to be purified can then be precipitated and equanimous, divergence behind any protein which is still soluble.The second method of purifying the IgG serum protein is ion exchange chromatography. This is a widely applied method of protein refining and uses positively budged groups or negatively charged groups immobilised onto a hydrophilic support, in this case DE- 52. Serum Proteins with an opposite net charge to that of the immobilised exchanger will bind to the column. Other serum proteins will pass through. Because the charge on proteins changes with pH, it is possible to attach a protein to the exchanger at one pH, then elute it by changing the cushion. Alternatively proteins can be eluted by passing an increasing concentration of salt through the column. The method works best for IgG which have high isoelectric points, at rough pH 8.6. This method can also be used how to separate different subclasses of IgG.Ammonium sulphate is less effective in the refining if IgG, but it is useful for the isolation of large IgM.Samples of each fraction will then be separ ated by cataphoresis on an agarose gel. Antibody will then be allowed to turn out towards the electrophoresed proteins from a trough cut gibe to the direction of electrophoresis. The proteins also diffuse from the positions they have reached after electrophoresis and precipitin arcs direct where antigen and antibody reach equivalent concentrations. This technique can be used to pick up whether a fraction contains any IgG and determine the degree of pollution of the IgG with other proteinsMaterials and MethodAmmonium Sulphate fractionation routine0.25 ml of saturated ammonium was added to 1ml of human serum, to produce a theme which 20% saturated with respect to ammonium sulphate.The rootage was mixed it was allowed to die hard in an ice for 15 minutes, and was centrifuged for 15 minutes at 1500 rotation per minute.The supported was poured and pallet was retained as fraction 1The supernatant from fraction 1 0.35 was added to bring to 35 % saturated with respect to ammoniu m sulphate the solution was left in an ice for 15minutes and it was centrifuged to recover the precipitate, the supernatant was poured in another tube while the pallet was retained as fraction 20.5 ml of saturated ammonium was added to the supernatant of fraction 2 to bring the solution to 50% , the solution was left in ice for 15 minutes to precipitate, it was centrifuged for 15 minutes the pallet was unploughed as fraction 3 while the supernatant containing 50% of protein was unbroken as fraction 4the absorbance of the fraction was measured at 280nmthe absorbance of 1mg/ml and 0.5mg of bovid serum albumin was measured was measuredBefore the immunologic analysis the fraction salt bailiwick were decreased by dialysis against buffer. 0.2(For more(prenominal) information refer to UEL hand out on protein purification)DE 52 ion exchange chromatographySerum provides was pre dialysed against 10mM trs/barbitone buffer pH 8.6 and chromatography column containing about 2mls of DE 52 w hich it has been equilibrated in the same bufferThe column was allowed to run until any superimposed buffer has run into the DE-52 gel avoiding the column to dryOuchterlony Double Diffusion AssayThe fractions collected from ion exchange chromatography were determined for the presence of IgG by using ouchterlony double diffusion method.The collected fractions were run against an anti-IgG antibody in an agarose gel. The revolve around well were filled with 3ul of anti-IgG and 3ul of the eluted fractions into the surrounding holes. Immunodiffusion was slowed to proceed for 24-48 hours an antigen-antibody precipitin line was observed.Single radial immune diffusionThis as a quantitative technique whereby the antigen is allowed diffuse from a well into a gel which contained its specific antibody, a precipitin will form when antigen concentration is check to the concentration of the antibody in the gel.ImmunoelectrophoresisMATERIALS AND METHODSPreparation of antigen parenthood samples w ere collected from ten clinically healthy cows using sterilized disposable needles (1.2 40 mm), clarified by centrifugation( speed of light0 g, 15 min) and diluted 11 with phosphate buffer saline (PBS,pH 7.2). then(prenominal) equal volumes of diluted serum and saturated ammoniumsulphate were mixed by slowly addition of the saturatedammonium sulphate solution with attractive stirring. aft(prenominal) centrifugation(1000 g for 20 min), the precipitate was water-washed twice with 50%saturated ammonium sulphate solution. The final precipitate wasdissolved in PBS followed by overnight dialysis against PBS. Proteinconcentration was quantified by a coomassie soil bindingassay (Bradford, 1976), using bovine serum albumin (BSA) as thestandard. Final protein concentration of solution adjusted to 1mg/mL.immunization of lapins with bovine immunoglobulinsThree hundred micro liters of prepared bovine immunoglobulins (1mg/mL) in PBS was emulsified with equal volumes of Freundscomplete adjuva nt (Sigma) and inoculated intramuscularly (I M) into leash 6-month-old New Zealand White rabbits. The rabbits were fedregular commercial diets. The second and deuce-ace inoculations wereperformed on days 21 and 35 with Freunds incomplete adjuvant(Sigma), and the ordinal inoculation was done on day 45 without anyadjuvant. After the final immunization, blood samples were takenfrom the rabbits and wareion of antibody was investigated bydouble diffusion and enzyme-linked-immunosorbent serologic assay tests.This study was approved by the Regional Medical Sciences searchEthics Committee of Tabriz University of Medical Sciences.Purification of rabbit anti-bovine immunoglobulinsImmunized rabbits sera were collected and precipitated by 50%ammonium sulfate. After dialysis against PBS and tris-Phosphatebuffer (40 tris and 25 mM phosphate, pH 8.2), ion-exchangechromatography was done on a DEAE-Sepharose fast scat (Pharmacia)in a laboratory made column at a flow rate of 0.25 mL/min.Protein concentration adjusted to 100 mg/mL and passed throughthe column. The column was washed in two go using Tris-Phosphate buffer for first washing step and Tris-phosphate buffercontaining100 mM NaCl for second washing step. The elutedproteins were collected in 5 mL fractions and analyzed by SDSPAGE.SDS-PAGE analysisThe purity of various IgG preparations was analyse using sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)under reduced conditions as described by Laemmli (Laemmli,1970). The final concentration of polyacrylamide solution was 13%.Samples were boiled with 2% SDS for 10 min and were loaded onthe electrophoresis gel. After separation, the proteins were stainedwith Coomassie Brilliant Blue G 250 (Blakesley and Boezi, 1977).Destaining was carried out in distilled water.Conjugation of rabbit IgG with peroxidaseThe conjugation was performed by the periodate method (Nakaneand Kawaoi, 1974) with some modifications. First, 4 mg of peroxidase(Sigma) was dissolved in 0.5 mL of distilled water in darkglasscontainer. Then sodium periodate (Merck) was added to thesolution, and the container was kept on a stirrer for 20 min at roomtempe-rature. The mixture was dialyzed against acetate buffer (0.1mM, pH 4.4) at 4C overnight followed by addition of 10 l ofcarbonate-bicarbonate buffer (0.2 M, pH 9.5). Eight milligrams ofpurified IgG in 1 mL of carbonate-bicarbonate buffer (10 mM, pH9.5) was add-ed to the active enzyme, and the container was put onthe stirrer. Then 150 l of fresh sodium borohydrate solution(Merck) was added to the above solution and was kept at 4C for1.5 h on the stirrer. The product was then dialyzed overnightagainst PBS at 4C and 1% BSA (Sigma) along with addition of0.01% sodium mirth-iolate (Merck).Enzyme linked immunosorbent assay (enzyme-linked-immunosorbent serologic assay)Direct ELISA was used to determine the titre of HRP conjugatedrabbit IgG against bovine immunoglobulins. 100 l of preparedbovine, sheep and goat immunoglobul ins, which was diluted 1100in PBS (10 g), was added to each well of a 96-well micro titer plateand incubated at 4C for 24 h. The wells were washed with PBSTween(0.05% Tween 20) three times and blocked with 200 l ofblocking solution (PBS-0.5% Tween 20). After a washing step, 100l of 1400, 1800, 11600, 13200, 16400 and 112800 dilutions ofprepared HRP conjugated anti-bovine immunoglobulins were addedto each well. The reaction was developed using 100 l of 3, 3, 5, 5-tetramethylbenzidine (TMB) as substrate and the absorbance wasdetermined at 450 nm after stopping the reaction by 5% sulfuricacid (Sigma).ResultsRESULTSProduction of rabbit anti-bovine immunoglobulinsIn order to survey production of antibody in rabbits andevaluating effectiveness of immunization, double diffusionand ELISA tests were performed. The titer of polyclonalanti-bovine IgG in double diffusion test was 8, whichappeared as a sharp band between antigen and antibodywells. The titer of anti-bovine immunoglobulins determ inedby ELISA was 16000.Purification of rabbit anti-bovine immunoglobulinsPurification of IgG rich fraction from immunized rabbitsera by ammonium sulfate precipitation followed byDEAE ion-exchange chromatography resulted in a highlypure fraction (first peak). The protein content of thisfraction was 45 mg which was about one third of primaryprotein content (Figure 1).SDS-PAGE analysisFigure 2 shows the results of SDS-PAGE for determiningthe purity of IgG, which was purified by ion-exchangechromatography. A distinct polypeptide band with molecularweight about 50 kDa agree to rabbit IgGheavy chains. The diffused bands between molecularweights of 20 30 kDa correspond to rabbit IgG lightchains. (Figure 2) The SDS-PAGE analysis showed thatpurification of IgG by ion-exchange chromatography resulted in a highly pure product. intelligenceThe purification of immunoglobulins presents severalpractical complications, especially for polyclonal antibodyproduction (Verdoliva et al., 2000). We used ionexchangechromatography for purification of rabbit IgGpolyclonal antibody. Separation and recovery of proteinsfrom ion exchange chromatographic media are affectedby factors such as buffer type and pH, aloofness of gradient,flow rate of the mobile phase, ionic strength and nature of counterpunch ion, and characteristic of the proteins. Theselection of ideal conditions for protein purification involveschanging some or all of these parameters(Tishchenko et al., 1998). This technique was wellestablished in our laboratory for purification of IgG antibody(Baradaran et al., 2006 Javanmard et al., 2005Majidi et al., 2005). Furthermore, ion-exchange chromatographyis considered as an economical alternative toaffinity and immunoaffinity chromatography. After purificationstep we obtained a protein with approximate purityof 98%. SDS-PAGE analysis showed that the protein withwell-nigh 50 kDa MW was rabbit IgG heavy chains.The light chain of rabbit IgG appeared as a diffused bandof 20 30 kDa molecular weights. It is likely that diffusedband of light chain could be related to different level ofdeglycosilation of protein during manipulation process.Conclusion