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Immunoassay is one of the most significant analytical methods that are used to detect and quantify of either antigen or antibody in a substance by means of their binding reaction method.
Antibody or antigen can be detected by attaching them with different types of labels materials such as enzyme, fluorescence and radioactive compound that will immerge a signal and analyse of interest can be measured.
Immunoassay with its highly affinity, specificity and sensitivity play an important role in the diagnosis of disease, monitoring humeral immune response levels and determining molecules of biological interest (Goldsby et al.,2003).
It is one type of immunoassay that was first discovered by Yalow and Berson in 1960. It was used to measure minute amount of insulin in the blood .Yalow awarded Noble Prize for the significance of this technique in 1977.This technique proved that some substances such as serum proteins ,hormones ,drugs and vitamins can be detected at very low concentration in the blood(Goldsby et al ,2003).
Disadvantages of (RIA).
It uses substances with radioactive material which could be affect human health.
It requires sophisticated and expensive equipments
Due to those problem, nowadays Enzyme linked immunosorbent assay (ELISA) is used instead of (RIA).
Different types of immunoassay:
Fluorescent immunoassay (FIA).
Enzyme immunoassay (EIA).
In our practical session we have used (EIA) or also known as Enzyme Link Immunosorbent Assay (ELISA), as it is being used largely in many clinical laboratories.
It was first introduced by Engval and Perlamann, Sscuurs and VanWeeman in 1971 (Werner et al. 2006).In our assay enzyme conjugated antibody or antigen are used to generate a coloured reaction product from colourless substances. There are different types of enzymes can be used in this type of assay but the most widely used are horse radish peroxidise and alkaline phosphatise.
Fig 1.0: Shows the principle of the Enzyme-Linked Immunosorbent Assay (ELISA) (Nairn R. 2002)
DIFFERENT TYPES OF ELISA TECHNIQUES:
This method is used to detect the amount of antibody that present in a sample. First antigens are coated with within the wells of microtitre plate. Then antibody is added to those wells and incubated to allow the binding of antibody- antigen reaction to take place. The plate is then washed to remove any unbound or excess antibody. Next, the plate is blocked with bovine serum albumin (BSA) to block any non specific binding of other antibody to the plate. A secondary enzyme linked antibody is added to the plate where it will be attached to the bound antigen- antibody. Then the plate is washed for a second time to remove any unbound secondary antibody. Finally the substrate is added which will change the colour from colourless to blue and the reaction mixture will be measured by spectrophotometer (the amount of colour present is directly proportional to the amount of antibody present).The detection of HIV antibodies viruses is done by this method (Thomas et al., 2007).
Fig 1.1: shows indirect ELISA method adopted from Goldsby et al, 2003.
This type of ELISA is used to either to detect or quantify soluble antigens. In this technique antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated-well. The more antigens present in the mixture the less antibody available to bind antigen coated well. As with other types of ELISA, enzyme-conjugated secondary antibody is added and can be used to determine the amount of primary antibody bound to the well as in an Indirect ELISA. (Elgert K. D 2009). In this type of assay amount of an unknown sample can be determined by its ability to compete with a labelled reference antigen for binding to an antibody attached to a plastic well. A standard curve is first plotted by adding various amounts of known unlabelled standard antibody and the assay then measures the antigens in the unknown samples by comparison with the standards.
Another type of ELISA known as a capture or sandwich ELISA can be used in the detection of antigen and secreted products such as cytokines. In this type of ELISA the antibody rather than the antigen is immobilised on the microtitre well i.e. rather than the antigen being directly attached to a plastic plate, antigen-specific antibodies are bound to the plate. These are able to bind antigen with high affinity and will therefore concentrate on the surface of the plate. A separate labelled antibody that recognises a different epitope to the immobilised first antibody is then used to detect the bound antigen (Janeway C.A et al 2001). The rate of enzyme action is directly proportional to the quantity of the enzyme-linked antibody present, which in turn is proportional to the amount of test antigen present.
Advantages of ELISA
It is highly specific
It is highly sensitive
It is more safer than (RIA) as it uses enzyme instead of radioisotope substance
It is easy to perform & requires cheap equipment
Antigens do not need to be purified before use.
Antibodies are immunoglobulines that react specifically with the antigen that stimulated their production. They make up about 20 % of the plasma portions.
Production of Monoclonal and polyclonal antibodies
Monoclonal antibodies are homogeneous immunoglobulin. The aim is to produce immortalized cells which only secrete immunoglobulin directed against the antigen used in immunization (Nairn, 2003). Monoclonal antibodies are produced by the offspring (clones) of a single hybrid cell, the product of fusion of antibody-producing plasma B cell and malignant antibody-producing myeloma cell. This technique is called hybridoma cells (Forbes et al., 2002).
Fig 1.4: Production of Monoclonal Antibodies from hybridomas cells illustrated from:
Polyclonal preparation result from the antibody response to immunization from infection in a host. In general, antibody to a specific agent is only a small fraction of the total antibody in a polyclonal preparation. Furthermore, polyclonal preparations usually contain antibodies to multiple antigens and include antibodies of various isotopes. Monoclonal antibodies preparations differ from polyclonal antibody preparations in that monoclonal antibody has one specificity and one isotope. As a result, the activity of monoclonal antibody preparation is considerably greater for the amount of protein present than polyclonal preparation also they are do not have lot variability associated with polyclonal preparation. Several monoclonal and polyclonal antibodies preparation are used for human therapy (Coico et al., 2003).
Fig 1.5: Production of polyclonal Antibodies: http://www.cytographica.com/overheads/antibody.html
Enzyme Linked Immunosorbentassay practical
Week 1, 2 practical's:
To determine the optimum antibody concentrations of two different types of antibodies provided (mouse-monoclonal antibody) and (goat polyclonal antibody) against rabbit antigen by performing a grid experiment.
Materials and methods:
Rabbit IgG antigen
Coating buffer: Phosphate Buffer Saline (PBS)
Wash buffer: 0.05% tween 20 in PBS, PH 7.4
Blocking buffer: 1% Bovine Serum Albumin (BSA)
Add 200 ul of rabbit IgG antigen in row A (A1-A12).
Add 100 ul of coating buffer (PBS) to all the remaining empty wells except those in row A.
Perform a serial dilution by taking 100 ul of rabbit IgG antigen from row A1 to B1 and gently mix by pipette.
Repeat step no 3 down to row G and then discard 100 ul from G1 Into a disposable container.
Repeat steps 3&4 to all the remaining columns (column 2-12).
Keep column (H) as a blank.
Cover the plate and incubate over night at 4'C.
Wash three times with wash buffer to remove any unbound antigen.
Block the plate with 1% bovine serum albumin (BSA) to remove any non- specific binding site.
Wash again and keep it ready for next week practical.
Fig1.4: illustrate week 1 practical
Materials and methods
Microtitre plate coated with rabbit IgG antigen (from 1st week practical)
Mouse anti-rabbit IgG (monoclonal antibody)
Goat anti-mouse IgG HRP conjugate (polyclonal antibody)
Goat anti-rabbit IgG HRP conjugate (polyclonal antibody)
Substrate solution (TMB)
Stop solution (1M HCL)
Wash buffer: 0.05% Tween 20 in PBS, PH 7.4
Diluent buffer: (PBS).
Leave the right half of the plate covered and untouched.
Add 100ul of diluents buffer (PBS) to all wells from column 2 to column 6
Add 200ul of monoclonal mouse anti-rabbit IgG (1/2000) to column 1 (A1 to H1)
Perform a serial dilution into each row by transferring 100ul of mouse anti-rabbit IgG from well A1 to well A2 and mix gently by pipette.
Continue the dilution till well A6 and discard 100ul of the mixture from well A6.
Repeat steps 4&5 to the remaining rows B to H.
Cover the plate and incubate it at room temperature for 30 minutes.
Cover the right side of the plate with a sticky plastic sheet.
Wash the left half of the plate three times with wash buffer.
Dry blot the plate after each washes using blotting paper.
Fig 1.5: illustrate from week 2 practical
To the right half of the plate add 100ul of diluent buffer (PBS) to all wells from column 8 to column 12.
Add 200ul of goat anti -rabbit IgG-HRP ( 1/2000) to column 7
Perform a serial dilution by transferring 100ul of goat anti-rabbit IgG- HRP (1/2000) from well A7 to well A8 and mix gently by pipette.
Continue the dilution till well A12 and discard 100ul of the mixture from well A12.
Repeat steps 3&4 to all the remaining rows (column 7 to column 12).
On the left half side of the anti-mouse IgG-HRP (1/5000) to all the wells from column 1 to column 6.
Cover the plate and incubate at room temperature for 30 minutes.
Wash the plate three times with washing buffer and then dry blot after each wash.
Add 100ul of TMB substrate to all the wells from column 1 to column 12 and incubate at room temperature for 10-15 minutes.
Observe the colour formation while incubating.(blue colour)
Add 50ul of stop solution (1ML HCL) to all the wells and observe the changes in colour from blue to yellow.
Read the plate on a plate reader at a wavelength of 450nm.
Fig 1.6: illustrate week 2 practical
Week 3, 4 practical's
To perform a sandwich ELISA method and determine the two unknown antigen concentrations(X and Y samples) using the optimum antibody concentration (that was obtained from previous practical's.
Materials and methods:
Mouse anti-rabbit IgG (1/2000)
Goat anti-rabbit IgG HRP (1/5000)
Patient Y sample
Patient X sample
Rabbit IgG 2ug/ml
Diluent buffer: phosphate buffer saline (PBS)
Wash buffer: 0.05% tween 20 in PBS, PH7.4
Blocking buffer: Bovine Serum Albumin (BSA)
Substrate: Sure Blue microwell peroxidase Contains 3, 3', 5, 5'-tetra-methylbencidine in an acidic buffer.
Stop solution :( 1ML HCL)
Add 100ul of mouse -anti rabbit IgG 1/2000 to columns (A1to H1 and A2 to H2) and to A3, A4, B3, B4.
Cover the plate and incubate it over night.
Wash the plate three times with wash buffer (PBS/Tween)
Block the plate with blocking buffer (BSA).
Wash the plate again with wash buffer and the plate is ready to use for next week practical.
To the coated microtitre wells with monoclonal mouse anti-rabbit IgG, add 200ul of rabbit IgG (2ug/ml) to wells A1 and A2.
Add 100ul of PBS to column 1 (B1-H1) and to column 2 (B2-H2).
Perform a serial dilution by transferring 100ul of rabbit IgG from well A1 to B1 and gently mix with pipette.
Continue dilution down till G1 and then discard 100ul from G1.
Repeat step 3&4 to column 2.
Add 100ul of unknown (sample X) to wells A3 and A4.
Add 100ul of unknown (sample Y) to wells B3 and B4.
Incubate the plate for 30 minutes at room temperature.
Wash the plate as usual 3X with PBS/Tween.
Dry blot after each wash using blotting papers.
Add 100ul of goat anti-rabbit IgG HRP (1/5000) to column 1, 2, 3 (A, B), 4 (A, B).
Incubate the plate for 30 minutes at room temperature.
Wash again 3X with PBS/Tween and dry blot after each wash.
Add 100ul of substrate (Sure Blue) to all wells used as mentioned above.
Cover the plate and incubate it at room temperature till the colour changes to blue.
Add 50ul of stop solution (1ML HCL) to the same wells and observe the yellow colour formation.
Finally read the plate using spectrophotometer at a wavelength 450nm.
Fig1.7: illustrates week 4 practical
Week 1 & 2
Absorbance @450 nm
Table 1.0: Shows the absorbance reading for the monoclonal mouse anti-rabbit IgG at different concentrations which was serially diluted and then taken at a wavelength of 450nm
Graph 1 : Shows the monoclonal mouse anti-rabbit IgG antibody titration curve. The curve was plotted by the
use of the results shown in Table 1.0 , of concentration of rabbit IgG (ng/ml) against the absorbance at 450nm
The graph1- above shows:-
Different dilution produce different mouse monoclonal antibody titration carves.
Dilution 1/4000, 1/8000 and 1/16000 showed high absorbance reading.
1/2000 dilution is more concentrated and the usage of antibody is high, it is not recommended commercially and will be excluded.
1/32000 and 1/64000 dilution have a poor linearity curve and are more diluted therefore excluded.
The dilution 1/4000, 1/8000 and 1/16000 showed high absorbance. But in case of the dilution 1/4000 is less diluted so the antibodies that are used are relatively high which mean large quantity is needed. This makes it commercially unacceptable.
1/8000 and 1/16000 Dilution are gradually and steadily increased which make them the optimum one, but compare between the two, in my opinion the optimum antibodies concentration is 1/16000 ng/ml because :
1. It is less expensive, commercially acceptable compare to 1/8000 dilution.
2. It has high absorbance and steadily increase which makes it reliable
3. It is more diluted than 1/8000 but still sensitive and able to detect almost same amount of the antibodies.
Absorbance @ 450nm
Table 1.1: Shows the absorbance reading for the polyclonal goat anti-rabbit IgG at different concentrations which was serially diluted and then taken at a wavelength of 450nm
Graph 2: Shows the polyclonal goat anti-rabbit IgG antibody titration curve. The curve was plotted by the
use of the results shown in Table 1.1 of concentration of rabbit IgG (ng/ml) against the absorbance at 450nm
Shows the absorbance reading of goat polyclonal labelled antibody in different dilutions.
From this graph:
In my opinion I think that the optimum antibody concentration is 1/4000 ng/ml, because it gave a good linearity curve and with high absorbance reading and it has difference absorbance reading in different concentration of rabbit IgG also it can detect the lowest concentration of the antigen.
1/2000 dilution shows high good increase of absorbance in the beginning but it then become stationary from dilution of about 500 dilution and above also because it is more concentrated and the consumption of antibody is high so it is excluded commercially.
1/8000 it start in the beginning with good linearity curve but it slow and did not rise further more so I think by compare with 1/4000 curve, 1/8000 is less in sensitivity and it is cannot detect concentration of high absorbance.
1/16000,1/32000 and 1/64000 dilutions, they all have a poor linearity curve and gave low observance concentration reading of rabbit IgG beside they are all more diluted and cannot detect concentration of high absorbance. According to all the above reasons they are excluded.
Absorbance @ 450nm
Table 3: Table shows the absorbance readings for the various concencentrations
of the rabbit IgG and also that of the unknown samples.
Graph 3: A standard curve for the rabbit IgG which will be used to determine the concentration of the unknown samples.
Graph 4: Shows the log concentration of rabbit IgG (ng/ml), this was done in order to determine the log concentration of unknown sample.
In order to get the exact concentration of the unknown samples a log10 graph was
plotted, and will then be calculated using the formula (y= mx - c )
X=concentration of unknwn sample
The mean absorbance= 0.685
Y=0.6079 x - 0.8861
0.685= 0.6079 x - .8861
0.8861 + 0.685= 0.6079 x
X =0 .6079/1.5711
X =387 anti log= ng/ml
The mean absorbance= 0. 029
Y = 0.6079 x - 0.8861
0.029= 0.6079x - 0.8861
0.029+0.8861 = 0.6079x
0.9151 = 0.6079x
X = 0.6079 / 0.9151
X = 0.664 anti log= ng/ml
The whole ELISA practical was run in four weeks time. During the first two weeks of the practical the aim was to determine the optimum antibody concentrations of both mouse anti-rabbit monoclonal antibody and goat anti-rabbit IgG HRP antibody by performing different dilutions using the indirect ELISA method.
In weeks 3 and 4, the aim was to find out the antigen concentrations of two unknown samples (X & Y) by plotting a curve with known concentration of sample as a standard using the sandwich ELISA method.
As a result of my practical work week 1 and 2 the curve obtained from monoclonal antibodies was good but the polyclonal was not perfect due to pipatting problem. The incubation time or temperature can also affect on the curve and this can be seen in the graph2. Monoclonal antibodies such as the mouse anti-rabbit IgG are antibody derived from single antibody-producing cells immortalised by fusion to a B- lymphocyte tumour cell line to form hybridoma clones The secreted antibody is momospecific in nature and therefore has a single affinity for a defined epitope. (Crowther 2001).
From week 3 & 4 the results of two unknown antigens were obtained by taking the mean absorbance readings of both test and samples and the curve was plotted.
There was also another way to find out the values of (X & Y) as mentioned in the results.
Moreover there are several problems that can interfere with ELISA results and should be taken under consideration:
Poor precision and standard curve and this may be due to:
Incomplete washing of wells (non specific reaction can take place as not all the non -bound label were removed).
Inadequate aspiration of wells (wrong or less amount of sample was transferred from well to well and this happened in my practical week2).
Unequal mixing of reagent.
Improper dilutions (in this test too much dilution was performed and may lead to false results if not paying attention).
Insufficient development of colour and this may be due to:
Wrong amount of substrate added.
Reduction in incubation time (this happened in our practical as it was reduced to 30 minutes).
Edge effect and this may be due to:
Irregular temperature around the working area (the temp around the edges of the plate is higher than in the middle of the plate).
Wrong fixing of the plate cover can lead to evaporation.
Reagent not kept in room temperature before use.
Antibody quality and concentration
There are several tips that can be done to improve the assay:
Use of multi channel pipette: this will reduce the chances of errors as well as reduces time consumption.
Use of automated plate washer as this will enhance the precision of the technique.
Make sure to incubate the plate at proper time and temperature (app 1 hr).
Overall the four weeks practical's was useful .from my practical work, I got the following results:
The optimal concentration of mouse-anti rabbit IgG monoclonal antibody was 1/16000 .
The optimal concentration of goat-anti rabbit IgG polyclonal antibody was 1/4000.
The X value concentration was approximately ng/ml.
The Y value concentration was approximately ng/ml.
In summary, ELISA is the method of choice because of its high affinity and specificity of antibody-antigen reaction, ease of performance, safe and cheap equipments are required. In future, I think more practice should be done with ELISA with the use of automated instrumentation and automated washer.