Ouchterlony Double Diffusion Assay Biology Essay

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Polyclonal antibodies are produced by different B- lymphocytes in response to the same antigen, which recognise different parts of the antigen. Because the human immune system cannot know in advance what pathogens it will 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 components of the immune system to destroy the target. These naturally-occurring polyclonal antibodies play a crucial role in triggering an immune response

Polyclonal 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 diffusion, radial immuno-diffusion, ammonium sulphate precipitation and ion exchange chromatography.


The aim of this practical is to compare the purification of serum IgG by ammonium sulphate and ion exchange chromatography.

The first purification step will normally involve a method such as fractional precipitation with increasing concentrations of ammonium sulphate. This method is not designed to achieve total purification, but to remove as much contaminant protein as possible whilst retaining all the protein of interest. Most proteins will precipitate from solution at high salt concentrations, but the salt concentration required to precipitate them varies considerably. Ammonium sulphate will 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 separate the serum proteins into four fractions. A fraction containing the serum protein to be purified can then be precipitated and collected, leaving 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 purification and uses positively charged 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 buffer. 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 about pH 8.6. This method can also be used how to separate different subclasses of IgG.

Ammonium sulphate is less effective in the purification if IgG, but it is useful for the isolation of large IgM.

Samples of each fraction will then be separated by electrophoresis on an agarose gel. Antibody will then be allowed to diffuse towards the electrophoresed proteins from a trough cut parallel to the direction of electrophoresis. The proteins also diffuse from the positions they have reached after electrophoresis and precipitin arcs form where antigen and antibody reach equivalent concentrations. This technique can be used to determine whether a fraction contains any IgG and determine the degree of contamination of the IgG with other proteins

Materials and Method:

Ammonium Sulphate fractionation Procedure

0.25 ml of saturated ammonium was added to 1ml of human serum, to produce a solution which 20% saturated with respect to ammonium sulphate.

The solution was mixed it was allowed to stand in an ice for 15 minutes, and was centrifuged for 15 minutes at 1500 rotation per minute.

The supernatant was poured and pallet was retained as fraction 1

The supernatant from fraction 1; 0.35 was added to bring to 35 % saturated with respect to ammonium 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 2

0.5 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 kept as fraction 3 while the supernatant containing 50% of protein was kept as fraction 4

the absorbance of the fraction was measured at 280nm

the absorbance of 1mg/ml and 0.5mg of bovine serum albumin was measured was measured

Before the immunological analysis the fraction salt content were reduced by dialysis against buffer. 0.2

(For more information refer to UEL hand out on protein purification)

DE 52 - ion exchange chromatography

Serum provides was pre dialysed against 10mM trs/barbitone buffer pH 8.6 and chromatography column containing about 2mls of DE 52 which it has been equilibrated in the same buffer

The column was allowed to run until any overlying buffer has run into the DE-52 gel avoiding the column to dry

Ouchterlony Double Diffusion Assay

The 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 centre 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 diffusion

This 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 equal to the concentration of the antibody in the gel.



Preparation of antigen

Blood samples were collected from ten clinically healthy cows using

sterile disposable needles (1.2 - 40 mm), clarified by centrifugation

(1000 g, 15 min) and diluted 1:1 with phosphate buffer saline (PBS,

pH 7.2). Then equal volumes of diluted serum and saturated ammonium

sulphate were mixed by slowly addition of the saturated

ammonium sulphate solution with gentle stirring. After centrifugation

(1000 g for 20 min), the precipitate was washed twice with 50%

saturated ammonium sulphate solution. The final precipitate was

dissolved in PBS followed by overnight dialysis against PBS. Protein

concentration was quantified by a coomassie dye binding

assay (Bradford, 1976), using bovine serum albumin (BSA) as the

standard. Final protein concentration of solution adjusted to 1


Immunization of rabbits with bovine immunoglobulins

Three hundred micro liters of prepared bovine immunoglobulins (1

mg/mL) in PBS was emulsified with equal volumes of Freund's

complete adjuvant (Sigma) and inoculated intramuscularly (I M) into

three 6-month-old New Zealand White rabbits. The rabbits were fed

regular commercial diets. The second and third inoculations were

performed on days 21 and 35 with Freund's incomplete adjuvant

(Sigma), and the fourth inoculation was done on day 45 without any

adjuvant. After the final immunization, blood samples were taken

from the rabbits and production of antibody was investigated by

double diffusion and ELISA tests.

This study was approved by the Regional Medical Sciences Research

Ethics Committee of Tabriz University of Medical Sciences.

Purification of rabbit anti-bovine immunoglobulins

Immunized rabbits sera were collected and precipitated by 50%

ammonium sulfate. After dialysis against PBS and tris-Phosphate

buffer (40 tris and 25 mM phosphate, pH 8.2), ion-exchange

chromatography was done on a DEAE-Sepharose fast flow (Pharmacia)

in a laboratory made column at a flow rate of 0.25 mL/min.

Protein concentration adjusted to 100 mg/mL and passed through

the column. The column was washed in two steps using Tris-

Phosphate buffer for first washing step and Tris-phosphate buffer

containing100 mM NaCl for second washing step. The eluted

proteins were collected in 5 mL fractions and analyzed by SDSPAGE.

SDS-PAGE analysis

The purity of various IgG preparations was checked using sodium

dodecyl 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 on

the electrophoresis gel. After separation, the proteins were stained

with Coomassie Brilliant Blue G 250 (Blakesley and Boezi, 1977).

Destaining was carried out in distilled water.

Conjugation of rabbit IgG with peroxidase

The conjugation was performed by the periodate method (Nakane

and Kawaoi, 1974) with some modifications. First, 4 mg of peroxidase

(Sigma) was dissolved in 0.5 mL of distilled water in darkglass

container. Then sodium periodate (Merck) was added to the

solution, and the container was kept on a stirrer for 20 min at room

tempe-rature. The mixture was dialyzed against acetate buffer (0.1

mM, pH 4.4) at 4°C overnight followed by addition of 10 μl of

carbonate-bicarbonate buffer (0.2 M, pH 9.5). Eight milligrams of

purified IgG in 1 mL of carbonate-bicarbonate buffer (10 mM, pH

9.5) was add-ed to the active enzyme, and the container was put on

the stirrer. Then 150 μl of fresh sodium borohydrate solution

(Merck) was added to the above solution and was kept at 4°C for

1.5 h on the stirrer. The product was then dialyzed overnight

against PBS at 4°C and 1% BSA (Sigma) along with addition of

0.01% sodium mirth-iolate (Merck).

Enzyme linked immunosorbent assay (ELISA)

Direct ELISA was used to determine the titer of HRP conjugated

rabbit IgG against bovine immunoglobulins. 100 μl of prepared

bovine, sheep and goat immunoglobulins, which was diluted 1:100

in PBS (10 μg), was added to each well of a 96-well micro titer plate

and incubated at 4°C for 24 h. The wells were washed with PBSTween

(0.05% Tween 20) three times and blocked with 200 μl of

blocking solution (PBS-0.5% Tween 20). After a washing step, 100

μl of 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 dilutions of

prepared HRP conjugated anti-bovine immunoglobulins were added

to each well. The reaction was developed using 100 μl of 3, 3', 5, 5'-

tetramethylbenzidine (TMB) as substrate and the absorbance was

determined at 450 nm after stopping the reaction by 5% sulfuric

acid (Sigma).



Production of rabbit anti-bovine immunoglobulins

In order to survey production of antibody in rabbits and

evaluating effectiveness of immunization, double diffusion

and ELISA tests were performed. The titer of polyclonal

anti-bovine IgG in double diffusion test was 8, which

appeared as a sharp band between antigen and antibody

wells. The titer of anti-bovine immunoglobulins determined

by ELISA was 16000.

Purification of rabbit anti-bovine immunoglobulins

Purification of IgG rich fraction from immunized rabbit

sera by ammonium sulfate precipitation followed by

DEAE ion-exchange chromatography resulted in a highly

pure fraction (first peak). The protein content of this

fraction was 45 mg which was about one third of primary

protein content (Figure 1).

SDS-PAGE analysis

Figure 2 shows the results of SDS-PAGE for determining

the purity of IgG, which was purified by ion-exchange

chromatography. A distinct polypeptide band with molecular

weight about 50 kDa corresponding to rabbit IgG

heavy chains. The diffused bands between molecular

weights of 20 - 30 kDa correspond to rabbit IgG light

chains. (Figure 2) The SDS-PAGE analysis showed that

purification of IgG by ion-exchange chromatography resulted in a highly pure product.


The purification of immunoglobulins presents several

practical complications, especially for polyclonal antibody

production (Verdoliva et al., 2000). We used ionexchange

chromatography for purification of rabbit IgG

polyclonal antibody. Separation and recovery of proteins

from ion exchange chromatographic media are affected

by factors such as buffer type and pH, length of gradient,

flow rate of the mobile phase, ionic strength and nature of

counter ion, and characteristic of the proteins. The

selection of ideal conditions for protein purification involves

changing some or all of these parameters

(Tishchenko et al., 1998). This technique was well

established in our laboratory for purification of IgG antibody

(Baradaran et al., 2006; Javanmard et al., 2005;

Majidi et al., 2005). Furthermore, ion-exchange chromatography

is considered as an economical alternative to

affinity and immunoaffinity chromatography. After purification

step we obtained a protein with approximate purity

of 98%. SDS-PAGE analysis showed that the protein with

approximately 50 kDa MW was rabbit IgG heavy chains.

The light chain of rabbit IgG appeared as a diffused band

of 20 - 30 kDa molecular weights. It is likely that diffused

band of light chain could be related to different level of

deglycosilation of protein during manipulation process.