Enzyme Linked Immunosorbent Assay Analysis Biology Essay

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Enzyme-Linked Immunosorbent Assay (ELISA). It depends on antibodies labeled with an enzyme react with their corresponding antigens in vitro to generate coloured reaction products in the presence of colourless substrate. There are variety of enzymes could be used in ELISA such as alkaline phosphatase, horseradish peroxidase and β-galactosidase (1).

At the end of the enzymatic reaction is measured by spectrophotometer at 450 nm and a reference filter of 620 nm.

ELISA is a qualitative test in which it utilizes a standard curve based on known concentrations prepared previously, so the unknown concentration is determined (1).

Antibodies to be used along with ELISA are polyclonal antibodies and monoclonal antibodies. The polyclonal antibodies are antibodies that can bind to different epitops on the antigen. They are a mixture of immunoglobulins secreted from B cells against a specific type of antigens. Mouse, rabbit and goat are typically selected animals to derive this polyclonal type of antibodies.

Monoclonal antibodies are mainly produced by immunizing a mouse to fuse the myeloma cells to produce this type of antibodies. They are highly specific in which they recognized only one epitop in an antigen.

There are many types of ELISA; they are direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA and ELISPOT (Enzyme-Linked Immunosorbent Spot). Multiplex ELISA is recently administrated to medical laboratories. The variation of these types depends on signal detection and labeling method (2).

In direct ELISA, directly labeled antibodies are used. While the target antigens are coated to the micro-well plate. It is quantitative type in which the end reaction product is read by colorimetric (3).

Although this type is relatively quik, but the antibodies themselves needed to be labeled each and every time and that is considered to be time consumption. It is noteworthy that by this direct ELISA we could avoid the problem of cross-reactivity of the secondary antibodies with test sample's antigens.

The second type is indirect ELISA, in this there are two steps used: first primary antibodies are incubated with the antigens. Then the labeled secondary antibodies are added. The secondary antibodies recognized the primary antibodies which and there are many labeled antibodies available commercially. It is good to mention that the primary antibodies are not affected by the labeling used for the secondary ones. One of its advantages is that indirect ELISA is sensitive because the secondary antibodies can be conjugated to many epitops in primary antibodies. Since there are two incubation periods, the first incubation is for the primary antibodies and the second incubation is for the secondary labeled antibodies, this type of ELISA is not as quick as direct ELISA (4). The next graph demonstrates the indirect ELISA principle.

Figure .1 www.uq.edu.com 2008

Competitive ELISA is the third type of ELISA technique. In which it is differ than the other ELISA forms. In it; a mixture of unlabeled antibodies with the target antigens is incubated. Then it is added to micro titer plate coated with antigens. If the mixture contains a lot of antigens, no enough antibodies will remain free to bind to the antigens in the coated plate. Then secondary labeled antibodies are added. At the end, substrate is added to activate the enzyme and end with a coloured reaction product (5).

Figure. 2 competitive ELISA (Roitt, 1991)

However; in this type of ELISA low absorbance means high level of target antigens and vice versa. Using un-purified primary antibodies consider as an advantage in competitive ELISA. There is a little problem associated with this type of ELISA that is if saturated mixture of antibodies added to a small amount of antigens, these antigens may not be detected. To overcome this, it is better to use slightly low antibodies mixture to reduce the binding sites.

Figure.3 sandwich ELISA (Roitt, 1991)

Sandwich ELISA is another common type of ELISA. In it two different types of antibodies are used which react with different epitops on an antigen. Antibodies of known concentration will be attached to the micro titer plate, and then the test sample with unknown antigen concentration is added. After the incubation period a washing process is carried out to remove any unbound antigens. Secondary antibodies labeled with enzyme will be added. A second washing is done to remove any extra secondary antibodies. Finally substrate is adding to generate a colour which could be then read by spectrophotometry (5). The above graph demonstrates the principle of sandwich ELISA method.

It is noteworthy that pregnancy test is one application of Sandwich ELISA. Usually monoclonal mouse antibodies are used to detect the HCG (Human Chorionic Gonadotropin) which present in pregnant ladies.

The last type of ELISA is ELISPOT. It is modified form of ELISA. It is a quantitative and qualitative and conceder to be sensitive ELISA. Either monoclonal or polyclonal antibodies coated to the micro plate. Then isolated lymphocytes and antigens are added and should be left for incubation. During this incubation period, the lymphocytes will be activated by the presence of antigens in the mixture. This will leads to cytokine secretion which will bind then to the coated antibodies. Cells and antigens are then washed away; secondary labeled antibodies will be added in the next step. A second washing is needed as well. Substrate is finally added to create a colour which can be measured as spots by ELISpot reader system (6).

Figure. 4 ELISPOT's well (WP exec PHR-2010)

The wells from ELISPOT test will appear like in the previous figure (7).

Practical procedures: (during all 4 weeks)

WEEK'S ONE practical was done to perform a grid experiment in order to determine the best concentration of the antibody by using mouse monoclonal anti-rabbit IgG and Goat anti-rabbit IgG peroxaidase conjugated.

In the first row A, add 200ul of rabbit IgG (2ug/ml).

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

Carry out serial dilution by transferring 100ul from (1-12) to (A-G) rows as shown below.

Figure .5 Serial dilution of rabbit IgG with PBS.

Discard the 100ul from G row and H row will be used as blank.

Incubate the plate overnight at room temperature.

Wash 3 times with PBS.

Block the plate with BSA.

Washed three times with PBS, dried and kept for week 2.

For WEEK'S TWO practical the 1st half of the plate will be used for monoclonal Abs while the 2nd half used for polyclonal Abs.

MONOCLONAL ANTIBODIES:

Add 200ul of monoclonal mouse anti-rabbit IgG.

Add 100ulof PBS to rest of wells starting from column 2 to column 6.

Dilute serially horizontally with 100 ul.(for all rows)

For column 6 the extra 100 ul should be discarded after the serial dilution.

Incubated the plate for 45 minutes at room temperature.

Figure .6 Serial dilution of monoclonal mouse anti- rabbit.

Wash 3 times with PBS.

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

Incubate at room temperature for 45 minutes.

Wash 3 times with PBS.

Add 100ul of substrate and wait for yellow colour development.

Add 50ul of 1 M HCL to stop the reaction.

Read the plate using the spectrophotometer at (450 nm).

POLYCLONAL ANTIBODIES:

Add 200ul of (Goat anti-rabbit IgG) to column 7.

Add 100ulof PBS to rest of wells remaining in the 2nd half of the plate.

Carry on with a serial Dilution with 100ul from (A7) till (A12); discard the extra 100ul from (A12).

Cover the plate and incubate for 45 minutes at room temperature.

Wash three times with PBS.

Add 100ul of substrate (TMP) to each well in the 2nd half of the plate.

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

Read the plate using the spectrophotometer at (450 nm).

Figure .7 Demonstration of goat anti-rabbit IgG-HRP dilution on the second part in week 2.

WEEK 3 &4: Sandwich ELISA was used to establish calibration curve and determination of 2 unknown samples.

METHOD:

Add 100ul of monoclonal (Mouse anti-rabbit IgG) to each well shown in the next figure.

Figure .8 monoclonal mouse anti- rabbit added to the wells.

Incubate overnight and then wash 3 times with washing buffer (PBS).

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

Wash 3 times with PBS and leave it to dry.

Week 4:

Figure .9 Distribution of rabbit IgG, PBS and X and Y.

As shown in above graph, 200ul of rabbit IgG (2ug/ul) was added in A 1 and A2 only.

Add 100 of PBS to the remaining wells in columns 1&2.

Dilute serially with 100ul from 1a to 1g as well as from 2a to 2g.

Row H will be used as blank.

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

Also add 100ul of unknown sample (Y) in duplicate to B3 & B4.

Incubate 30 minutes at room temperature.

Wash plate 3x with PBS/Tween.

100ul of goat anti-rabbit IgG-HRP was added to all wells.

Incubate for 30 minutes at room temperature, and then wash 3 times.

Add 100 ul of substrate to all wells used and the microplate was covered to protect it from light, then it was incubated at room temperature, and the color change was observed by nicked eyes.

Stop the reactions by adding 1M CLH.

Read at 450 nm.

RESULTS:

From practicals in week 1&2:

Monoclonal antibodies:

Concentration

ng/ml

1/2000

1/4000

1/8000

1/16000

1/32000

1/64000

2000

1.862

1.82

1.714

1.544

1.169

1.032

1000

1.401

1.27

1.131

1.091

0.838

0.628

500

0.674

0.621

0.617

0.573

0.5

0.339

250

0.4

0.342

0.33

0.311

0.213

0.169

125

0.171

0.157

0.126

0.113

0.095

0.062

62

0.074

0.07

0.063

0.056

0.045

0.029

31

0.032

0.028

0.026

0.025

0.016

0.013

0

0

0

0

0

0

0

Figure .10 Monoclonal antibodies absorbance with different concentrations.

Figure .11 Monoclonal antibodies standard curves.

Polyclonal antibodies:

Concentration

ug/ml

1/2000

1/4000

1/8000

1/16000

1/32000

1/64000

2000

3.301

2.703

1.773

0.99

0.524

0.278

1000

2.732

2

1.176

0.605

0.291

0.154

500

2.4

1.56

0.806

0.432

0.227

0.116

250

2.03

1

0.708

0.356

0.19

0.086

125

1.49

0.738

0.44

0.232

0.113

0.048

62

0.953

0.446

0.233

0.138

0.064

0.035

31

0.514

0.226

0.073

0.03

0.027

0.019

0

0

0

0

0

0

0

Figure .10 Monoclonal antibodies absorbance with different concentrations.

Figure .13 Polyclonal antibodies standard curves.

From practicals in week 3&4:

absorbance

0.24

0.239

0.175

0.176

0.27

0.21

0.087

0.085

0.222

0.19

0.203

0.148

0.148

0.108

0.1

0.08

0.064

0.049

0.025

0.024

Figure .14 Absorbance of STDs, X and Y.

Y = 50

X = 328

Figure .15 Rabbit IgG curve of known concentration.

Figure .16 Shows the log plot of the absorbance obtained of rabbit IgG, which displays a straight line from where the unknown samples can be calculated using the equation created in this graph.

Discussion:

This ELISA practical was divided into two parts. Fristly, week one and week two practical was done to obtain the best concentration of both polyclonal and monoclonal antibodies. In this experiment indirect ELISA was applied to generate series of standard curves for each one of the titration used. Figure. 11 which showed different dilutions of monoclonal antibodies. It also gives us that 1/8000 is the best concentration because it just not increases sharply which is the case for 1/2000 and 1/4000 dilutions. In addition, 1/2000 and 1/4000 had some technical errors at around 500 ng/ml of rabbit IgG. 1/8000 needs low amount of antibodies as well which is not costly affective.

On the other hand; 1/16000, 1/32000 and 1/64000 curves are showing low absorbance values and they are not reflecting the optimum concentration that we were looking for during this practical.

According to figure. 13 the best concentration of polyclonal antibodies to be used is 1/4000. From the figure again; the 1/4000 curve was distributed evenly and raised in a rhythmic way. It will give a good variation of concentration, too. Since 1/2000 took high amount of polyclonal antibodies comparing to 1/4000, I didn't go for it because it is quite expensive. There is another problem affecting 1/2000 curve that is it could be splitting apart. The first part is sharply increased and then in the second part it gave steady line which will affect the patients' results. Moreover the remaining curves are not suitable to be utilized because they gave low readings.

Then in week 3 and week 4, the experiment was carried out to estimate the concentrations of two unknown samples X and Y. Both X and Y was run in duplicate to get the mean absorbance. X and Y concentration was finally obtained from the curve in figure 15 which showed standard curve of known concentration of rabbit IgG. Although the X and Y concentration confirmed by the equation which is generated from figure 16.

The results of X and Y will be like the following:

X= 0.151

The equation: Y=0.1119x -0.1307 Y means X's absorbance

0.151= 0.1119x In con. -0.1307 con. Means concentration

0.151 + 0.1307 = 0.1119x In con.

0.2817= 0.1119x In con.

In con. = 0.2817/0.1119

= 2.517

Anti In (2.517) = X's con.

X concentration = 328 ng/ml

Y= 0.0615

The equation: Y=0.1119x -0.1307 Y means Y's absorbance

0.0615= 0.1119 x In con. -0.1307

0.0615+0.1307= 0.1119x In con.

0.1922= 0.1119 x In con.

In con.= 0.1922/0.1119

= 1.7

Anti In (1.7)= Y's concentration

Y concentration = 50 ng/ml

ELISA Optimization:

After all we have to consider that there are several parameters affecting the ELISA results, such as:

Incomplete washing of wells as well as inadequate aspiration of well and unequal mixing of reagents are all factors lead to poor precision.

Poor standard curve due to improper dilution and incomplete washing which is done to remove any unbound antibodies.

Inadequate colour development. This could be interfere with many factors like:

Inadequate volume of substrate was added to the wells.

Short incubation period.( we have shorten it regarding to our class time)

The room temperature was not optimum. 25 ° C is required to perform ELISA.

Edge effect usually because of irregular temprature in the working place or evaporation of the test due to inadequate fixing of plate cover and reagent not kept at room temprature.

Edge Effects,this phenomenon discribes the variation of ODs in the well's edage and the wall of the central region of the plate (8). Also the tempreature could be varied between the well's edage and the center area of the well.

Evaboration may affect the ELISA and to over come this we have to cover the micro plate tightly.

To conclude, the evaluation of the practical done can be marked as fair good results as more practice is needed for the ELISA technique. Also the usage of more automated instruments will influence the work done in the practical.

Finally, it is expected that ELISA is going to show more and more variations in its techniques; as we applied the indirect ELISA for weeks one and two and for week three and four sandwich ELISA was performed because it is very reliable and good test that scientist is putting more effort to improve it day by day.

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