Immuno Assays Played Vital Role In Disease Diagnosis Biology Essay

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Immunoassays have played a vital role in disease diagnosis, monitoring the level of the humoral immune response and identifying molecules of biological or medical interest. These assays are important in regulatory work because of their sensitivity, rapidity and specificity.

The antigen antibody interactions are similar to an enzyme substrate interaction apart from not leading to an irreversible chemical alteration in either parts, because these interaction are so specific, immunoassays are needed to detect their presence.

Antigens are substances that when introduced into an animal will cause the production of antibodies. Antibodies are y shaped proteins found in sera which are produced as a response to a specific antigen. They have different classes and molecular weights, are composed of two identical heavy chains and two identical light chains, and connected via disulphide bonds.

In this experiment Polyclonal antibodies were used and these are produced by injecting animals with antigen (adjuvant) to increases immunogenicity, so that the animal can produce an immune response. Antibodies are then secreted from its blood. The antibodies produced this way are mainly immunoglobulin G (IgG) molecules which make up about 10% of the protein in the serum. IgG can be purified from blood serum by different methods but the two methods used here are a) ammonium sulphate precipitation and b) ion exchange chromatography. The first purification method used is ammonium sulphate precipitation in order to separate the serum proteins into fractions depending on the concentration of the ammonium sulphate needed to precipitate them. Each fraction will then be assayed for IgG content and total protein content.

Ammonium sulphate precipitation ((NH4)2SO4) is one the oldest methods and most commonly used for purifying antibodies. The use of ammonium as the precipitating salt gives the advantage of high solubility that is not totally dependent on temperature. At 50% saturation a higher yield of IgG at lower purity is obtained but smaller fragments of the molecule need higher salt concentrations for preparation.

Ion exchange chromatography is the method to be compared with ammonium sulphate precipitation. This method is extremely useful for the separation of proteins and isolation of immunoglobulin. Proteins are bound to an ion exchange matrix with an opposite charge and the level to which a protein binds depends on its charge density. Proteins are eluted by increasing the ionic strength of the medium and by altering the pH (as the pH of the buffer approaches the isoelectric point of each protein, the net charge becomes zero and so the protein no longer binds to the ion exchanger. CM-cellulose and DEAE-cellulose are the most common. However, these two methods on their own cannot obtain purity they must be combined with other methods.

Other immunoassays will then be used to test purified fractions and these are a) Ouchterlony double diffusion assay, b) Single Radial Immunodiffusion (SRID) and c) Immunoelectrophoresis.

Ouchterlony Double Diffusion, in this technique the antigen and antibody are allowed to migrate towards each other in a gel and a line of precipitation is formed where the two reactants meet, its position is determined by the concentration of the antigen and antibody in the agar. There is an advantage to this assay because several antigens can be compared around a single well of antibody or antigen, so this is a qualitative method.

SRID, in this technique as the antigen diffuses radially a ring of precipitation forms around the well and moves outwards, eventually becoming stationary at equivalence. When equivalence is reached the diameter and area of the ring are related to the antigen concentration in the well but as it diffuses out concentration falls. Quantitative measurements are given from the precipitin lines.

Immunoelectrophoresis is a powerful analytical technique with a great resolving power, combining prior separation of antigens by electrophoresis with immunodiffusion against and antiserum. It is used to identify proteins in complex mixtures, which are separated by electrophoresis in an agarose gel. The electrophoresed protein will diffuse in all directions from the point to which it has moved and antibody will diffuse sideways. If the antibody recognises the antigen in the sample, a precipitin line will form. By using agarose gel, proteins are able to diffuse through pores.

AIM

The aim of this experiment is to remove as much IgG protein as possible whilst keeping all or most of the IgG, by using the various methods (immunoassays) in this experiment and then compare the two main purification methods to assess the purified fractions and their efficacy on the process overall.

MATERIALS

Human Serum DE-52 cellulose

Saturated ammonium sulphate solution Dialysed Serum

Centrifuge 0.5M NaCl

10mM Tris/ Barbitone buffer pH8.0 2M NaCl

Spectrophotometer Ouchterlony plate

1mg/ml Bovine Serum Albumin (BSA) Distilled Water

Micro Dialysis Chamber Whole Serum

pH 8.6 buffer Bromophenol blue

Chromatography Column

0.5mg/ml, 0.2mg/ml, 0.1mg/ml and 0.05mg/ml IgG

Electrophoresis tank

SRID (Agarose slides with Rabbit anti human IgG)

METHODS

As per schedule

The first step was to use ammonium sulphate precipitation method to separate the serum proteins into four fractions by salting out. Here as salt concentration increases less water is available for hydration of protein, so proteins come together.

A 20%, 35% and 50% ammonium sulphate saturation solution was made and four pellet fraction produced from them. These four fractions were redissolved in 0.5ml 10mM Tris/ Barbitone buffer pH 8.0

The protein content of each fraction was measured by an absorbance reading at 280nm. A measurement was also taken by a 1mg/ml and a 0.5mg/ml solution of Bovine Serum Albumin to standardise the assay.

Salt content in fraction was reduced by placing fractions to dialyse ‘a la Ray’.

The second step was to use the DE-52 ion exchange chromatography to separate the serum proteins, based on their different charge, in pH 8.6 buffer. At this pH the charge on IgG is neutral while most proteins have a negative charge, therefore, IgG will pass through the column whereas other serum proteins will bind.

0.5ml of dialysed serum was pipetted into the column and fractions of 1ml eluate were collected.

Tris/Barbitone buffer was eluted from the column and 10 fractions were collected.

A small sample from each column fraction was run against an anti IgG antibody in an agarose gel (Ouchterlony Double Diffusion Assay), a qualitative method to identify fractions containing IgG.

One fraction was made out of the fractions containing IgG, by mixing them all in one tube.

The same volume of IgG solutions with different concentrations were pipetted into an Agarose gel (SRID) with Rabbit anti human IgG antibody.

Samples of each fraction were pipetted into an electrophoresis agarose gel (Immunoelectrophoresis) with whole serum and bromophenol blue and put in a electrophoresis tank.

CAUTION- Do NOT lift the lid up of the gel tank whilst running.

RESULTS

Fig1. Ammonium sulphate fractions reading for protein content @280nm.

Fractions

Absorbance @ 280nm

1

0.161

2

0.294

3

0.175

4

0.205

5

0.440

1mg/ml BSA

0.739

0.5mg/ml BSA

0.253

Fig2. Results for protein concentration and total amount of protein for each fraction, based on the calibration graph

Fractions

Protein Concentration (mg/ml) x dilution factor (10)

Protein concentration x 0.5mg (dialysed serum = Total amount of protein (mg/ml)

1

2.1

1.05

2

3.7

1.85

3

2.4

1.2

4

2.7

1.35

5*

5.75

17.25

*Fraction 5 is a mixture of 3 fractions containing IgG and to get the total amount it was multiplied by 3 to get the total amount of protein.

Fig3. Results table for the Single Radial Immune Diffusion assay with respective diameters.

Standards mg/ml)

Square ring diameter (mm)

Square ring diameter (mm2)

0.05

8.76

17.5

0.1

7.70

59.3

0.2

4.19

76.7

0.5

0

0

Fractions

Square ring diameter (mm)

Square ring diameter (mm2)

At what dilution factor?

1

6.0

36

1 in 20

2

7.30

53.3

1 in 20

3

0

0

0

4

0

0

0

5

6.92

47.9

1 in 5

Fractions

Square ring diameter (mm2)

IgG concentration x dilution factor

1

43.2

2

53.3

3

none present

none present

4

none present

none present

5

47.8

Ouchterlony Diffusion plate

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