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B cells participation to adaptive immunity by producing antibodies and the restraint of B cells to an immunogenic is often gauge by analyzing the definite antibody produced in a humoral immune response which found in the blood or plasma.(Janeway. Et al , 2005)
Antibody molecules are much definite for their corresponding antigen, could discover one molecule of a protein antigen out of more than 10 8 similar molecules. They are many techniques use the specificity and stability of the antigen binding by antibodies ( antibody assay or serological assay ). ( Janeway. Et al, 2005).
Primary interaction assay is used to measure the direct binding of the antibody to antigen.
Secondary interaction assay is used to measure the amount of antibody present by the change it induce in the physical state of the antigen ( e.g. like the precipitation of soluble antigen or the clumping of antigenic particles ).
Affinity chromatography: In this method, specific antibody can be isolated from an tiserum which exploits the specific binding of antibody to antigen held on a solid matrix.
Figer 1 ( Kenneth M. et al. 2008 )
Radioimmunoassay ( RIA ), Enzyme - Linked Immunosorbent Assay:
RIA and ELISA are direct binding assay for antibody or antigen and both work on the sample principle, but the means of detecting specific binding is different.
Radioimmunoassay ( RIA ): is commonly used to determine the levels of hormones in blood and tissue fluids. Its need a pure preparation of a known antigen or antibody, or both. Its used radioactively labeled usually with I 125.
Enzyme-Linked Immunosorbent Assay ( ALISA ):
ALISA is used to determines the amount of an antibody or an antigen are presence in the sample. The principle of ELISA is depends on an enzyme Synchronous with an antibody reacts with a colorless substrate to produce a colored reaction product. They were different type of substrates can be used in ELISA like alkaline phosphate, horseradish peroxidase, and β-galactosidase. ELISA is more sensitivity, safety, and less costly. Antigen or antibody can be determine in qualitative and quantitative in different method of ELISA.(Thomas J.K et al, 2007).
By this method antibody can be determine in serum or other sample . The antigen is coated in microtiter plate well. After that any free antigen removed and the micro titer plate is washed. Sample having antibody is added allowed to react with antigen attached to the well then removed the excess antibody and washed. Then addition of secondary antibody which contains the enzyme conjugates. This will bind to the primary antibody. Then wash again to remove the free secondary antibody followed by addition of substrate of the enzyme. Finally by using spectrophotometric plate reader, the coloured produced is estimated. (Thomas J.K et al, 2007)
Figure1: Indirect ELISA (Thomas J.K et al, 2007)
Antigen can be measured by a sandwich ELISA. By this method the antibody is coated on a microtiter plate wells and a sample containing antigen is added to react with antibody. Then the plate is washed and the sconed antibody ( enzyme-linked antibody ) is added to react with the bound antigen. After that the plate is washed and substrate is added to produce the colored reaction and the plate is ready to measured. (Thomas J.K et al, 2007).
Figure2: Sandwich ELISA (Thomas J.K et al, 2007)
Competitive ELISA :
It is another method for the determination of antigen as antibody is incubated with the antigen from the test sample. Then this mixture is added to a micotiter well where antigen is coated. So if the sample contains many antigens less free antibody will be available to bind to the antigen in the well. After that, addition of enzyme conjugated secondary antibody which will react with primary antibody. Finally measurement of the coloured produced. If the concentration of antigen is higher, the absorbance is lower. (Thomas J.K et al, 2007)
Figure5: Competitive ELISA (Thomas J.K et al, 2007)
1- 96 well microtiter plate.
2- Adjustable micropipette.
3- Plate reader at 450nm.
Materials and Reagents :
1 Coating buffer : PBS
2 Wash buffer: 0.05% Tween 20 in PBS, pH 7.4
3 Diluent: PBS
4 Antigen: rabbit IgG
5 Coating antibody: mouse monoclonal anti-rabbit IgG ( to be used determined on weeks 1 and 2 ).
6 Detection antibody: Goat anti-rabbit IgG-Peroxiase conjugated ( dilution to be used
determined on weeks 1 and 2 ).
7 Colour reagent ( TMB ).
8 Stop solution ( ClH 1M ).
For the preparation of antibody titration for the monoclonal and polyclonal antibodies the whole plate will be used.
Week 1 and 2: ( Titration of antibodies to be used in the sandwich ELISA )
1- 100ul of coating buffer is added to wells from row B to row H to the microtiter plate.
2- 200ul of rabbit lgG antigen ( 2000ng/ml ) is added to row A, mix well.
3-100ul from row A is added to row B and mix well.
4- The dilution is continued until row G and discarded 100ul from row G.
5- Row H is kept as control.
N.B. The concentration of rabbit lgG antigen is as follow:
Row A ( 2000ng/ml )
Row B ( 1000ng/ml )
Row C ( 500ng/ml )
Row D ( 250ng/ml )
Row E ( 125ng/ml )
Row F (62ng/ml )
Row G (31ng/ml )
Row H ( control )
5- The plate is incubated overnight at room temperature.
6- The plate is washed with buffer (This step was done by the technical lab staff ).
7- The plate is blocked by using 1% bovine serum albumin/BSA in phosphate buffer saline/PBS (This step was done by the technical lab staff).
8- The plate is washed with buffer and dry (This step was done by the technical lab staff).
9- The plate is divided into two part:
A- The first part is for monoclonal antibody titration.
B- The second part is for polycolonal antibody titration ( should be cover and untouched)
10- 200ul of monoclonal mouse anti-rabbit lgG ( 1/2000 ) is added to column No.1 in the first part of the plate.
11- 100ul of PBS is added from column 2 to column 6.
12- Mouse anti-rabbit lgG is diluted by transferring 100ul form well no. 1 of row A to well no. 2 of the same row and mix well.
13- The dilution is continued until well no.6 and discarded 100ul from it.
14- Repeat this presses up to row H.
N.B. The concentration of mouse anti-rabbit lgG is estimated by dilution and the concentration of rabbit lgG antigen is as follow:
Column 1 (1/2000 )
Column 2 (1/4000 )
Column 3 (1/8000 )
Column 4 (1/16000 )
Column 5 (1/32000 )
Column 6 (1/64000 )
15- The plate is incubated for 45 minutes at room temperature (Incubation period was reduced from one hour due to the shortage of practical time)
16- The plate is washed three times with buffer and dried.
17- At the second part of the plate ( Polycolonal titration antibody ), 200ul of the goat anti-rabbit lgG- HRP is added in column no.7.
18- 100ul of PBS is added from column 8 to 12.
19- 100ul of goat anti-rabbit lgG-HRP is transferred from well 7 to well 8 in row A and mix well.
20- Repeat this presses to row H and discarded 100ul form it.
N.B. The concentration of goat anti-rabbit lgG is estimated by dilution and the concentration it is as follow:
Column 7 (1/2000 )
Column 8 (1/4000 )
Column 9 (1/8000 )
Column 10 (1/16000 )
Column 11 (1/32000 )
Column 12 (1/64000 )
21-100ul of perosidase enzyme ( goat anti-mouse lgG ) is added to the first part of the plate
22- The plate is incubated for 45 minutes at room temperature. (Incubation period was reduced from one hour due to the shortage of practical time)
23- The plate is washed three times with buffer and dried.
24- 100ul of the substrate is added to all wells on the plate and waited to colour change to light blue.
25- 50ul ClH 1M is added to stop the reaction to all wells on the plate and yellow coluor is came.
26- Read the plate reader ( Dynex Technologies ) at 450nm.
Weeks 3 and 4:( ELISA calibration curve and determination of 2 unknown samples)
100ul of antibody monoclonal anti-rabbit IgG is added to column 1, column2 and wells A and B of column 3 and 4.
Incubate the plate at room temperature overnight.
Wash them with buffer. (This step was done by the technical lab staff).
Block the plate with Bovin Serum Albumine. (This step was done by the technical lab staff).
Wash them again with buffer. (This step was also done by the technical lab staff).
Keep the plate dried for the next week practical.
1 - 200 ul of rabbit IgG (2ug/ml) is added to the first row wells of row A in column
1 and 2.
2 - 100ul of PBS is added to column 1 and 2 expect row A.
3 - 100 ul is transferred from row A to row B and continue the pressers until the G
row for column 1 and 2.
4 - 100ul of unknown sample X is added to row A in column 3 and 4.
5 - 100ul of unknown sample Y is added to row B in column 3 and 4.
6 - Cover the plate to avoid the effect of the light in the solution.
7 - Incubate the plate for 30 minutes at room temperature.
8 - Wash the plate with buffer 3 times and dried.
9 - 100ul of goat anti-rabbit IgG HRP in all used wells.
10 - Incubate the plate for 30 minutes at room temperature.
11 - Wash the plate with buffer 3 times and dried.
12 - 100ul of substrate (TMB) to all the used wells.
13 - When the colour developed stop the reaction by the addition 50 ul of 1M HCL
14 - Read the plate reader ( Dynex Technologies ) at 450nm.
15 - Draw the graph to find out the concentration of unknown X and Y.
The graph shows the absorbance at 450nm at y axis of the Mouse anti-rabbit IgG monoclonal antibody titration against the concentration of rabbit IgG ( ng/ml) at x axis in different dilution ( 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:62000 ).
The graph has six line graph,one of the mean things to nota is the highest dilution ( 1:64000 ) have the lowest absorbance ( absorbance is directly proportional to the concentration ).
Another things which is shown from the graph is that all six curve are increasing but is different levels. Line graph of 1:64000 shows staedy increase up to 0.8 absorbance , than its slightly increase up to 1.2 absorbance. Dilution of 1:32000 and 1:16000 have almost the same results, with a little higher of 1:16000 dilution. They have a moderated rise from 0 absorbance to 1.8 absorbance.
Dilution group 1:2000, 1:4000,and 1:8000 were the highest, from 0 to 1250 they shown the same results about 1.6 absorbance where dilution 1:4000 have the highest 2.186 and dilution 1:8000 was the seconded and 1:2000 was the 3 th.
Theoretically dilution 1:2000 should have the highest absorbance but due to the personal error it became number three in order.
In conclusion, the fast there dilution are good whereas dilution 1:4000 is the past. but a lot of antibodies will be consumed which will make them expensive assays. Therefore, the best dilution which shows reasonable absorbance was 1:8000 dilution. This dilution can detect the lowest concentration of antigen and also can be used for more numbers of samples comparing with smaller dilutions. When developing ELISA, it should be as cheap as possible since the antibody is expensive. Therefore, the suitable assay is the one which can distinguish between different concentrations and should be also more diluted which ultimately will be cheaper and more economic.
The graph shows the absorbance at 450nm at y axis of the Goat anti-rabbit IgG HRP polyclonal antibody titration against the concentration of rabbit IgG ( ng/ml) at x axis in different dilution ( 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:62000 ).
The graph has six line graph, one of the mean things to nota is that the highest dilution ( 1:64000 and 1:32000 ) have the lowest absorbance ( absorbance is directly proportional to the concentration ).
Another things which is shown from the graph is that all six curve are increasing but is different levels. Line graph of 1:64000 and 1:32000 shows slightly increase up to 0.5 absorbance. Dilution of these two are not suitable as they can not detect higher absorbance and also their interruptions of results will not be accurate. With a little higher absorbance of 1:16000 dilution. Dilution 1:8000 and 1:4000 have a moderated rise from 0 absorbance to 1.8 absorbance but 1:4000 was a higher than 1:8000.
1:2000 was the higher and have a dramatic increase from 0 to about 2.7 absorbance.
The best dilution which shows reasonable absorbance was 1:8000 dilution. This dilution can detect the lowest concentration of antigen and also can be used for more numbers of samples comparing with smaller dilutions. When developing ELISA, it should be as cheap as possible since the antibody is expensive. Therefore, the suitable assay is the one which can distinguish between different concentrations and should be also more diluted which ultimately will be cheaper and more economic.