Reagents Of Elisa Have Long Shelf Life Biology Essay

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ELISA is generally the simple and high sensitive immune assay that combine the specificity of antibodies with the sensitivity of simple enzyme assays. This method was described in 1971 by Engvall, Perlman, schuurs and VanWeemen. It can give a valuable measurement of antibody or antigen concentration. ELISA techniques use substrate that generate soluble products. An enzyme conjugated to an antibody and reacts with a colourless substrate which will generate a coloured reaction product which will be absorbed and reported by taking the reading with specially multichannel spectrophotometer (Crowther, 2001).

This type of ELISA utilizes the technique of directly labeling the antibody itself. Solid surface (microwell plates) are coated with a sample including specific antigen. When specific antigen added it will bind to the labelled antibody, after washing the enzyme labelled antibody added and only bind the specific antigen that captured (Crowther, 2001)

Advantages:

Quick test since one antibody is utilized.

Eliminate cross-reactivity with the second antibody.

Disadvantage:

Every antibody needs to be labelled which is time consuming and expensive.

Cannot chose primary antibody tag from one assessment to another.

Few signal magnification.

Reduce primary antibody immunoreactivity due to labelling.

Figure 1: diagram shows direct ELISA assay. Adopted from [http://www.genwaybio.com/gw_file.php?fid=6056].

The Indirect ELISA:-

This method is used to determine the amount of antibody that is present in the sample. The microwell plate of this method is coated with labeled antibody and captured antigen as indirect assay. Then it goes in incubation time and this allow the secondary labeled antibody to attach and identify the first antibody (ABBAS et al., 2003) .

Advantage:

large selection of labelled secondary antibody are commercially available.

Flexible.

The primary antibody will be not effected by labelling.

More sensitive than direct technique.

Disadvantage:

Non specific signal may occur due to cross-reactivity with the secondary antibody.

Time consuming due to extra incubation time.

Figure 2: diagram shows indirect ELISA assay. Adopted from [http://www.genwaybio.com/gw_file.php?fid=6056].

Sandwich ELISA:-

This method measures the amount of antigen between 2 layers of antibodies and it can be called captured method (Janeway et al., 2005). The antigen that should be measured it must include at least two antigenic sites, so it will be able to bind with the two different antibodies and act as sandwich technique. Either monoclonal or polyclonal antibodies used as the capture and detecting antibodies in this method. The antibodies attached to the plate well, and sample antigen is added, so it will be bind to the antibody after incubation. The unbound antigens are removed by washing. Then detecting(primer)antibody added to bind specific antigen. Secondary enzyme- linked antibody added and bind the primary antibody. After incubation, the unbounded antibodies removed by washing and the substrate added, that will react and form colour measured by spectrophotometer.

The sensitivity of sandwich ELISA depends on 4 main factors:

Molecules number of the primary antibody that are attached to the solid surface.

The avidity of the primary antibody for the antigen.

The avidity of the secondary antibody for the antigen.

The definite action of the secondary antibody.

Figure 3: diagram shows sandwich ELISA assay. Adopted from [http://www.genwaybio.com/gw_file.php?fid=6056].

Competitive ELISA:-

This method can be a choice when two ''matched pair'' antibodies are not exist for a target . Unlabelled antibody is incubated with the antigen. This bound antibody antigen mixture are then added to an antigen coated well. Then the plate will be washed and antibody that has been bind to coated antigen will stay only and the unbound antibody removed. An enzyme linked antibody is added to the mixture. Finally substrate will be added and the remaining enzyme will draw out chromogenic or fluorescent signal (wild, 2005). In interpretation, the higher original antigen concentration the weaker the eventual signal.

Figure 4: diagram shows competitive ELISA assay. Adopted from [http://www.genwaybio.com/gw_file.php?fid=6056].

multiplex ELISA:-

Multiplex ELISA can be found in different types and techniques and some of them are in the research field to develop them as soon as possible .Several types of primary antibodies can attached on glass plate to capture matching antigens in a biological specimen like plasma, tissue extract or cell lysate (Murphy et al., 2008).

Figure 5: diagram shows multiplex ELISA assay. Adopted from [http://www.genwaybio.com/gw_file.php?fid=6056].

2.0 Production of Polyclonal and Monoclonal antibodies:-

Monoclonal antibody: are monospecifc similar antibodies that are produced by identical immature cells which all are clones of a unique parent cell. Monoclonal antibodies produced by the combination of myeloma cells with the mice spleen cells that has immunized with the desired antigen. Then it will be fused into special media which will allow them to grow. Next the hybrid cells are chosen and cloned. Monoclonal antibodies are identical and particular to the interested epitope. (Chapel, et al., 2006) The main advantage of this application, produce virtual unlimited quantities of a particular antibody.

Figure6. The diagram shows the principle of Monoclonal antibody production. Adopted from [http://www.proteopedia.org/wiki/index.php/Monoclonal_Antibody]

Polyclonal antibody: represent antibodies that are taken from multiple B cells sites. They are a mixture of immunoglobulin molecules that are secreted against particular antigen, each recognize a different epitope. They are produced by injecting mammals such as rabbit, goat and horse with an antigen. This stimulates the B lymphocytes to make IgG immunoglobulins specific for the antigen. Then the polyclonal antibodies separated from the mammal's serum. This variety of antibodies allow the researchers to identify various epitope sites on the intrested protein . However polyclonal antibody has a number of disadvantages (Luttmann, et al., 2006), and they are:-

Difficult to produce from batch to batch

Produce a huge amount of non specific antibody.

Multiple epitopes increase the percentage of cross reactivity.

Figure 7. The following diagram shows Principles of polyclonal antibody production. Adopted from [http://www.biotech.iastate.edu/facilities/hybridoma/SERVICEpAb.htm].

Enzyme Linked Immunosorbent Assay practical

Practical work, Week (1):

Materials and Reagents required:

96-well microtiter plate.

Adjustable micropipette.

Rabbit IgG Antigen.

Coating antibody: mouse monoclonal anti-rabbit IgG.

Detection antibody: Goat anti-rabbit IgG- Peroxidase conjugated.

Diluent and Coating Buffer: - Phosphate Buffer Saline (PBS).

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

Colour reagent (TMB substrate).

Stop solution (ClH 1M)

In the first week we will prepare the dilution of monoclonal and polyclonal antibodies by using Sandwich ELISA technique, we will use the whole 96-microplate and divide it into two halves. The first part will be monoclonal antibodies and the second part will be polyclonal antibodies

Procedure:-

Add 200ul of rabbit IgG antigen in the microtiter plate row A from (1to12).

Add 100ul of coating buffer (PBS) in the remaining empty microtiter plate wells.

Then do serial dilution by taking 100ul of the rabbit IgG antigen from A1to A12

Repeat the dilution for the entire remaining column by the same way in previous step.

Discard the 100ul of the rabbit IgG antigen after column (G) and keep column (H) as a BLANK.

Cover the plate and incubate it at overnight.

Wash the microtiter plate with PBS three times, blocked with 1% Bovine Serum Albumin/BSA in.

Wash again and was kept ready to use for week 2 practical.(steps 7&8 were done for the student by the lab technician).

Figure 8 Demonstrates the week 1 procedure:-

Practical work, Week (2):

Column (1-6) in the microtiter plate were used for mouse anti rabbit IgG antibody (monoclonal antibody), and the second part from column(7-12) were used for Goat anti-rabbit IgG- HRP (polyclonal antibody).

Add 100ul PBS Diluent from column (2A to 6A) and repeat this step for the other rows (B to H).

Add 200ul of the mouse anti rabbit IgG antibody (1/2000) monoclonal antibody to the column in the microtiter plate from column A1 to column H1.

Dilute serially 100ul of (Mouse anti Rabitt IgG) from row (A to H) horizontally from column (1 to 6) and column 6 discard extra 100ul.

Incubate the plate for 45 minutes at room temperature.

Wash the microtiter plate with PBS washing solution 3 times, Before washing remember to cover the remaining wells from 7- 12 with special sticky sheet that been given to you.

Add 100ul of goat anti-mouse IgG- HRP(fixed dilution 1/5000) to all wells in the first half.

Incubate the plate for 45 minutes at room temperature.

Wash the microtiter plate with PBS washing solution 3 times.

100 ul of substrate (TMP ready to use) to all wells and wait till the reaction takes place and blue color will developed.

Stop the reaction by adding 50 ul of ClH 1M and the color will be changed from blue to yellow.

Finally, read your plate by using spectrophotometer at 450nm with reference of 620nm.Then you can draw your graph.

Figure 9 Demonstrates of monoclonal mouse anti rabbit IgG antibody dilution in week 2

The second part of the practical is to do goat anti rabbit IgG (polyclonal) antibody

First of all remove the cover from wells (7 to 12), then Add 200ul of goat anti rabbit IgG- HRP(1/2000) Polyclonal antibody to column (A7 to H70.

Add 100ul of PBS Diluent buffer to all remaining wells.

As we done before do serial dilution by taking 100ul from well (A7 to A12) horizontally, remember to discard the extra 100ul from A12.

Repeat step 3 to all remaining wells in the second half of the microtiter plate.

Cover the microtiter plate and Incubate for 45 minute, and then wash it with washing buffer three times.

Add 100ul of the substrate to all wells.

Stop the reaction by adding 50ul of stop reagent ClH 1M to all wells.

Finally read your microtiter plate by using spectrophotometer at (450 nm) with reference of (620 nm), and now you can draw your graph.

Figure 10 Demonstrates the polyclonal goat anti rabbit IgG antibody dilution in week 2

Practical work, Week (3):

Add 100ul of capture antibody (monoclonal Mouse anti-Rabbit IgG in the microtiter plate column 1 from (A1 to H1), column 2 from (A2 to H2), A3, A4, B3 and B4.

Cover the plate with special sticker that have been provided to you and incubate the plate overnight.

Wash the plate three times with washing buffer PBS.

Add 1% Bovine Serum Albumin to the plate to block it.

Wash the plates again with washing buffer PBS for three times.

The plate is ready now to be used in week four practical.

N.B steps from (3 to 6) were done for students by laboratory technician.

Figure 11. shows the demonstration of week 3 to know the concentration of unknown samples.

Practical work, Week (4): continue the sandwich ELISA

Add 200ul of rabbit IgG ( 2ug/ml) to well A1 and A2.

Add 100ul of PBS diluent from well (B) to (H) in number 1 and 2.

Dilute serially 100ul in column 1 and 2 starting from row A till row G, then Discard the extra 100ul from well (G1 and G2).

Wells H1 and H2 will be used as BLANK.

Add 100ul of unknown sample X in (A3 and A4),and add 100ul of in unknown sample Y in (B3 and B4)

Incubate 30 minutes in the room temperature, however the correct time of incubation is one hour but it was reduced to 30 minutes because of the time shortage.

Wash the microtiter plate three times with the washing buffer PBS.

Add 100ul of goat anti rabbit IgG-HRP labelled to all the used well in the microtiter plate.

Incubate for 30 minutes in the room temperature, however the correct time of incubation is one hour but it was reduced to 30 minutes because of the time shortage.

Wash again with washing buffer PBS for three times.

Add 100ul of substrate (TMB sure blue) to all used wells cover it with dark paper for 10 minutes and wait till the wells generate blue colour.

Stop the reaction by adding 50ul of the reaction stop reagent HCl, then the colour will be changed from blue to yellow. Cover the microtiter plate and read it by using spectrophotometer at (450 nm) and the reference filter of (620 nm).

Figure 12. shows the demonstration of week 4

Result:-

Table (1) show the absorbance and concentration of monoclonal antibody (WEEK 1&2)

Rabbit IgG

1/2000

1/4000

1/8000

1/16000

1/32000

1/64000

2000

1.946

2.186

2.107

1.829

1.586

1.144

1000

1.688

1.646

1.651

1.455

1.278

0.962

500

1.181

1.156

1.159

1.111

0.893

0.824

250

0.788

0.719

0.693

0.661

0.482

0.395

125

0.322

0.403

0.338

0.32

0.215

0.178

62

0.179

0.175

0.159

0.138

0.095

0.082

31

0.082

0.075

0.062

0.06

0.035

0.03

 

0

0

0

0

0

0

 

 

 

 

 

 

 

Monoclonal antibody

Graph (13) illustrate table no 1;

Table (2) shows the concentration of polyclonal antibody (WEEK 1&2)

Polyclonal antibody

Rabbit IgG

1/2000

1/4000

1/8000

1/16000

1/32000

1/64000

2000

0.976

0.723

0.451

0.237

0.125

0.062

1000

0.913

0.587

0.33

0.166

0.09

0.045

500

0.766

0.458

0.263

0.11

0.063

0.029

250

0.579

0.337

0.164

0.08

0.042

0.02

125

0.382

0.217

0.104

0.047

0.026

0.01

62

0.23

0.115

0.056

0.025

0.014

0.005

31

0.118

0.06

0.032

0.008

0.009

0.001

 

0

0

0

0

0

0

Graph (14) illustrate table 2

Table (3) shows the results of week 3& 4

Concentration

Abs 1

Abs2

Average

Average-average blank

Sample X

Sample Y

Average X Average Y

Average X,Y

- Average Blank

2000

0.325

0.273

0.299

0.2775

0.183

0.095

0.169

0.1475

1000

0.295

0.258

0.2765

0.255

0.155

0.076

0.84

0.265

500

0.278

0.222

0.25

0.2285

250

0.233

0.189

0.211

0.1895

125

0.150

0.131

0.145

0.1235

62.5

0.101

0.089

0.095

0.0735

31.25

0.065

0.052

0.0585

0.037

0

0.02

0.023

0.0215

Graph 15 shows the standard curve of Rabbit IgG

Standard curve of Rabbit IgG

Absorbance 450 nm

Rabbit IgG

From the above graph we can determine the value of X & Y.

The X value is 177 and the Y value is 44

However there is another way to find out the value of sample X and sample Y and that by Log Plot as the graph and calculation that shown below

Graph 16 shows the log plot to find out the concentration of unknown samples

The equation that I have worked on is;

Y=0.1416x- 0.1706

For the unknown sample X:

Absorbance mean is 0.169

0.1475= 0.1416x -0.1706

0.1475+0.1706= 0.1416x

0.3181 = 0.1416x

x =0.3181/0.1416

x=2.25 Anti log 2.25 =178 Sample X = 178

For the unknown sample Y:

Absorbance mean is 0.0625

0.0625=0.1416x-0.1706

0.0625+0.1706=0.1416x

0.2331 = 0.1416x

x = 0.2331/0.1416

x=1.65 Anti log 1.65 = 45 Sample Y = 45

Discussion:-

The aim of the practical week 1 & 2 is to find out the best optimum concentration of the monoclonal and polyclonal antibody by using Sandwich ELISA Assay. The monoclonal antibody was done in the first part (1 to 6) of the microtiter plate and polyclonal antibody was done in the second part (7 to 12) of the microtiter plate.

For week 1 we used mouse anti rabbit IgG monoclonal antibody in column (1 to 6). Figure (13) shows the different curves for different dilution. As it shown in the graph dilution (1:2000) gives sharp increase, but it will be excluded because of the low concentration and high usage of antibody that make it unsuitable commercially and expensive.

However the dilution (1:4000) and (1:8000) shown very good and sensible reading, but it still not that good as it gives low concentration and more antibody are needed. On the other hand (1:16000) it gives the same result but it needs less antibodies than others which make it good commercially and cheap in the same time.

In dilution (1:32000) and (1:64000) they both are not showing reproducing the optimum concentration as they are more to be liner curves, so they will be excluded.

In constant from my results I suggest dilution (1:16000) is the best optimum concentration for monoclonal antibody, as it needs less antibody and it will be more cheaper and more significant to use in medical laboratories .

From table 2 that demonstrate in graph (14) also show different curves for different dilutions used in week 2 to find out the optimum (goat-rabbit IgG). As we can see from the graph, dilution (1/2000) show good curve but with low concentration and high usage of antibodies so it will be excluded. Dilution (1/16000), (1/32000) and (1/64000) will be excluded as they show liner curve. On the other hand, the dilution (1/4000) and the dilution (1/8000) show good curves with good reading.

In my opinion, dilution (1/4000) is the best optimum polyclonal antibody concentration as it needs low amount of antibody, that makes it cheap and commercially recommended.

The practical in week 3 and 4 were done to determine the concentration of unknown from two samples (X and Y) by using unknown concentration of antibodies to draw standard calibration curve. As you can see in the graph (15) we have to get the mean for (X and Y). graph (15) show the approximately concentration of the unknown (X and Y) samples. We can get the specific concentration of the unknown samples (X and Y) by applying the logarithmic graph as shown in graph (16). by that we can get the equation that can help us to calculate the exact concentration of unknown samples

The concentration of sample Y = 45 ng/ml

The concentration of sample X = 178 ng/ml

There are lot of small causes that may interfere with our results and may occur in any ELISA technique.

Errors in the practical and possible causes

Poor Precision.

Improper washing of the microwell plate (unbound antibody cause non specific reaction)

insufficient aspiration of the wells (antibody were mixed in different wells)

Unequal mixing of the reagent.

Poor standard curve.

Improper dilution of the highest standard or blank.

Improper washing.

Insufficient aspiration of the wells.

Inadequate Color development.

Inadequate volume of substrate has been added to the wells.

Shortage of incubation time.

Conjugate or colour reagent failure.

Edge effects.

Irregular temperature in the working area.

Wrong fixing of plate cover can lead to evaporation.

Reagent not kept in room temperature.

There are some useful precautions to avoid any problem in ELISA technique:-

Utilize multi-channel pipettes because it will minimize mistakes and save the time.

Utilize automated plate washer, because it will reduce contamination of the other wells with reagents while washing.

Incubation period should be for one hour, so the reaction can take place properly.

Use the specify quantity of reagent as its recommended by the manufactures, in the mean while keep the reagent in the recommended temperature before using it.

Cover the plate properly and block it after adding the antigens to avoid the non-specific reaction.

Conclusion:-

ELISA is a diagnostic technology that utilize the antigen-antibody compound and enzymes. And it becomes the most used technique nowadays in the medical laboratory due to the safety and accuracy. In addition, ELISA is cheap commercially and high-quality instrument for diagnosis of various diseases. Good results can be achieved with highly accuracy while applying as the manufacture recommended.

Overall, four weeks practical sessions were useful. However, I think more practice should be done and more advanced pipettes, plate washer and more incubation time may help to improve the quality of results.

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