Enzyme Linked Immunosorbent Assays Biology Essay

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Enzyme- linked immunosorbent assay also known as an enzyme immunoassay, which is a biochemical technique used to detect the presence of antibody or antigen by detecting the density of colour which is caused by enzyme-substrate reaction. ELISA technique is used in different fields, it is used in diagnostic tests for medicine e.g. detection of HIV antibodies in patient serum or for hormonal studies and other application. Moreover it is used as quality control in industries. In ELIZA an enzyme conjugated to an antibody reacts with a colourless substrate to generate a coloured reaction product. An antigen is immobilized in microtitre plate, and then specific antibodies applied so that it can bind to the antigens, then washing step to remove the unbound antibody. Finally, chemical substrate is added which reacts with the enzyme and convert it into a detectable signal.

Prior the development of enzyme immune assay, in 1950s two scientist "Yallow and Berson" discovered a technique in which they used radioactive substances and they were awarded Noble prize in 1977 for their discovery as they developed Radioimmunoassay (RIA) for insulin. Nevertheless, despite the specifity and the sensitivity of the test, it was costly, required special equipment and the most important defect it held potential threat due to reactive substances. Because of the health threat the RIA possessed, safer substitute was sought. In 1971 two scientists from Sweden published methods of ELISA. In ELISA an enzyme in alternate to radioactive signal is used which reacts with chemical substrate and causes colour change.

A number of variations of ELISA have been developed, allowing detection and measuring quantity of either antigens or antibodies.

Types

Direct ELISA

In this method, microwell plate is coated with the sample which contains the target antigen. Directly labelled antibody binds to the coated antigen, and the binding of labelled antibody is quantified by colorimetric.

Indirect ELISA

In this method, antigen is coated to the microtitre well and blocked, to prevent inferior protein to attach to well, next an unlabelled antibody added and bindsto the antigen. After this step washing is done to remove excess or unbound antibody. Next step, labelled antibody conjugated with enzyme is directed against the first antibody to react, and again washing step to remove the excess labelled antibody. After that, is to add substrate which reacte with conjugated enzyme and as a result colour will be produced which is left to be until it reaches the optimum reaction. Finally, the reaction is stoped usually using one molar hydrochloric acid and the colour is read by spectophotometre.

Sandwich ELISA

In sandwich ELISA, antigen level is detected between two layers of antibodies,however, the target antigen must have two epitopes atleast to act as sandwich"in order to bind with two deffirent antibodies". This method requires specific antigen antibody. First step, to add the specific antibody into microtitre well plate and then to add the antigen wich will bind to the antibody.second step, to wash away excess amount of antigen and add an enzyme linked antibody (secondary antibody) to the antigen-antibody complex in the microplate well. So the antigen will be captured between the two antibodies or it will be like a sandwich, hence the name Sandwich ELISA given. After washing substrat is to be added and is going to react with the enzyme and give the coloured signal which then could be read by a spectrophotometer.

Competitive ELISA

In this assay, mixture of antigen/antibody "which is antibody incubated in solution with a sample which contains antigen" is added to an antigen coated in microtitre well. If there is more more antigen present in the sample then a less amount of free antibody will be available to bind to the coated antigen. Next step, an enzyme conjugated secondary antibody specific for the isotype for the primary antibody bound to the well as in indirect ELISA. Nevertheless, in this assay the higher the concentration of antigen in original sample the lower the absorbance.

ELISA Reverse method and device (ELISA -R m&d)

A new technique in which a solid phase "made up of immunosorbent polystyrene rod with 4-12 protruding ogives" is used. Device entirely immersed in the test tube which contains the test substance.

Dipping of the ogives in the microwells of standard plate pre filled with reagents the following steps are carried out (washing, incubation in conjugate and incubation in chromogenous).

ELISA Practical

Materials:

Coating buffer:PBS

Wash buffer:0.05% Tween 20®in PBS, pH 7.4

Diluent buffer:PBS

Antigen : rabbit IgG

Coating antibody: mouse monoclonal anti-rabbit IgG (dilution to be used determined on weeks 1& 2)

Detection antibody: Goat anti-rabbit IgG-Peroxidase conjugated (dilution to be used determined on weeks 1&2)

Colour reagent

Stop solution (ClH 1M)

1% Bovine Serum Albumin (BSA) blocking buffer

96-well microtiter plate

Adjustable micropipette

Week 1 & 2

Aim:

To achieve a grid experimental to detect the optimal detection and capture antibody titration, by using monoclonal mouse anti-rabbit IgG and polyclonal goat anti-rabbit IgG antibodies.

Method:

In week one, we divided the plate into two parts. One half (columns 1-6) was used for monoclonal mouse anti-rabbit IgG antibody, and the second half (columns 7-12) was kept for polyclonal goat anti-rabbit IgG antibody.

Week One:

Add 200ul of rabbit IgG (2ug/ml) in the first row A from (1-12).

Add 100ul of PBS diluents in the rest of the wells.

Dilute serially 100ul in column (1-12) from A-G. (As shown in diagram 1).

Discard the extra 100ul from G row.

Row H will be used as blank.

Incubate overnight at room temperature.

Wash three times with PBS.

Blocked with 5% Bovin Serum Albumin for two hours at room temperature.

Washed three times with PBS, dried and kept for week 2. (Steps 6, 7 and 8 were done for the student).

Week two:

First half of the plate will be used for the monoclonal antibody, columns (1-6)

Add 200ul of monoclonal (Mouse anti-rabbit IgG 1:2000) to column 1.

Add 100ul of PBS diluents to the rest of the wells from column 2-6.

Dilute serially 100ul of (Mouse anti-rabbit IgG) from row A horizontally from column (1-6).(As shown in diagram2)

In column 6 the extra 100ul must be discarded after the dilution to keep the volume equal in all wells.

The same procedure in step (3&4) should be done with rows B, C, D, E, F, G and H.

Incubate the plate for 45 minutes at room temperature. (The optimum time is one hour, but it was reduced to 45 minutes due to shortage of time in the practical).

Wash the plate three times with buffer.

Add 100ul of (goat anti-mouse IgG) linked with HRP enzyme to all wells in the first half.

Incubate the plate at room temperature for 45 minutes. (The optimum time is one hour, but it was reduced to 45 minutes due to shortage of time in the practical).

Wash the plate with buffer and dry the plate.

Add 100ul of substrate to all wells and wait till the colour is developed.

Stop the reaction with 50ul of 1M HCl.

Read the plate using the spectrophotometer at (450 nm) with reference of (620 nm).

Finally draw the graph using absorbance results

The second half of the plate will be used for the polyclonal antibody.

Use the second half of the plate (columns 7-12)

Add 200ul of (Goat anti-rabbit IgG) linked with HRP enzyme to column 7.

Add 100ul of PBS diluents to the rest of the wells in the second half.

Dilute serially 100ul from (A7) horizontally till (A12), and discard the extra 100ul from (A12). (As shown in diagram 3).

The same should be done as in step (3&4) with rows B, C, D, E, F, G and H.

Incubate for 45 minutes at room temperature. (The optimum time is one hour, but it was reduced to 45 minutes due to shortage of time in the practical).

Wash three times with buffer.

Add 100ul of substrate (microwell Peroxidase substrate (1-componenet yellow)).

Stop the reaction by adding 50ul of 1M HCl to all wells.

Read the plate with spectrophotometer at (450 nm) with reference of (620 nm).

Draw the graph using absorbance.

Week Three: Sandwich ELISA for standard curve

Add 100ul of monoclonal (Mouse anti-rabbit IgG) (γ-chain specific) to the wells. (As shown in diagram 4).

Incubate overnight.

Washed three times with PBS.

Blocked with 5% Bovin Serum Albumin for two hours at room temperature.

Washed three times with PBS, dried and kept for week 4. (Steps 3, 4 and 5 were done for the student).

Week four: continue sandwich ELISA.

Add 200ul of standard (Rabbit IgG 2ug/ml, γ-chain) to A1 & A2 wells.

Add 100ul of PBS diluents to wells B till H in column 1 & 2.

Dilute serially 100ul of (Rabbit IgG 2ug/ml, γ-chain) in column 1 and 2 till G row.

Discard the extra 100ul.

Row H will be used as blank.

Add 100ul of unknown sample (X) in duplicate to A3 & A4 wells.

Add 100ul of unknown sample (Y) in duplicate to B3 & B4 wells. (As shown in Diagram 5).

Incubate the plate at room temperature for 30 minutes.

Wash the plate three times with buffer.

Add 100ul of (Goat anti-rabbit IgG) Linked with HRP enzyme in all wells used.

Incubate for 30 minutes at room temperature.

Wash the plate three times with buffer.

Add 100ul of substrate (TMB) to all wells.

After the colour development, stop the reaction with 50ul of 1M HCl.

Read the plate by spectrophotometer at (450 nm) with reference of (620 nm) and draw your graph using the absorbance reading.

Using your graph find out the results of X & Y samples.

Results:

Table (1) shows the result of monoclonal

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