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Immunology meaning is the study of the immunity that defence mechanism of the body against any disease or infection and those studies has response of organisms to foreign substances such as viruses, bacteria, and bacterial toxins. Immunoassay is an essential technique for diagnostic infection or disease in a patient.
Immunoassay was discovered in 1941 by Albort Coons and Collaborators they were used antibodies conjugated with fluorescein to localize cellular antigen. Well, these fluorescent molecules have the ability to absorb and release light at different wavelength (kindt, et al., 2007).
The immunoassay is a quantitative and qualitative analytical technique that depends on a reaction between antibody and antigen (Jane way, et al., 2005).
There are several types of immunoassay technique and they differ in specificity, sensitivity and rapidity but share in the mainly steps for preparing antibodies to capture antigen on the patient sample:
1. Enzyme linked immunosorbent assay (ELISA)
2. Radioimmunoassay (RIA)
3. Fluorescence polarization immunoassay (FPIA)
4. Cloned enzyme donor immunoassay (CEDIA)
5. Enzyme-multiplied immunoassay Technique (EMIT)
8. Chemiluminescent immunoassays
9. Particle immunoassays
2.0 Antigen and Antibodies
Antigen is a substance that when introduced into the body stimulates the production of an antibody and include toxins, bacteria, blood cells and viruses. Whereas, antibodies is a Y- shaped protein produced on the surface of B cells (B lymphocyte) to antigenic stimulus (Accetta D and Taunto PC, 2007).
3.0 Monoclonal Antibodies
In 1975 George Kohler and Cesar Milstein were able to discover a technique for monoclonal antibodies preparation. Monoclonal antibody is an increase specific antibody produced in the vitro (in the laboratory) in response to an antigen, which is detected and neutralized by that specific antibody. This antibody is specific for one epitope in antigen (Chapel et al., 2006).
Plasma cells from spleen are fused with cancer cells that generating hybrid cells this known is hybridoma technology. These cells now having the characteristics of the B cells with antibody type and cancer cell with the ability of growing continuously. Hybridoma cells secrete large amount of monoclonal antibodies (recognize one epitope) and can be cloned or grown from single colonies and investigated for specific antibody (Kindt., et al, 2007).
Figure 1: illustrates artificial production of monoclonal antibodies, adapted from (Michink S W, Sidhu S S, 2008).
4.0 Polyclonal Antibodies
Most antigens have multiple epitopes on them, and produce antibodies will be different depending on the antigenic epitope. This information was used to prepare polyclonal antibodies. Polyclonal antibodies are antibodies produced by different B lymphocytes to recognize a different epitopes region on the antigen. These antibodies usually produced by of a mouse, rabbit or goat. These animals injected many times and from time to time blood sample investigate for concentration of antibodies. When antibodies concentration reaches a determined amount, the antibodies recovered from the animals and purified (Barker K, 2005).
Figure 2: illustrates the production of polyclonal antibodies. Adopted from (Roland J, 2002)antibody purification
ELISA method became rapidly accepted now and is the most common enzyme immunoassay used to measure the concentration of antigens or antibodies in vitro. Eliza method is used in various areas like for screening blood donor, hormone level, auto-antibodies, syphilis, detection drug s and measurement toxin in contaminating food (kindt., et al, 2007). There are several principles used in ELISA such as Sandwich ELISA, Indirect ELISA, and Competitive ELISA.
5.1 Sandwich ELISA
In ELISA sandwich assay can be called capture method; fixed quantity of antibody is immobilized to a plastic wall of microtiter plate. Then, add the sample containing the antigens to the well which allow them to bind to antibody. The unbound antibodies are removed by washing followed by addition of enzyme-linked (horse-radish peroxidise or alkaline phosphatase) antibody that will bind to different epitopes on the antigens. After that the plate is washed again to remove the unbound antibody. As the antigen is present in between two antibodies the compound is called sandwich. Substrate e.g. TMB is added and the coloured reaction product is measured by a spectrophotometer. The test is run along with known standard concentration to help in comparison and calculation of antigen in the sample. This ELISA is fast and accurate. However, the disadvantage here is that not all antibodies can be used. (Abbas, A.K., et al, 2003).
Figure 3: illustrate Sandwich ELISA method. Adapted from (http://newenglandbiolabs.de/en/images/stories/cst_elisa_gross.jpg)
5.2 Direct ELISA
The direct ELISA technique is directly labelling antibody method. The target antigen on the sample is coated with microwell plate. After washing, enzyme-labelled- antibodies are added. Then after incubation and washing, the substrate is added. The mixture incubated for sometimes to allow colour develop. The disadvantages of this method are some antibodies are not suitable to direct labelling, time consuming and expensive (Crowther R J, 1995).
The Indirect ELISA assay is used to measurement the level of antibody in the sample. That sample is added to the wells, which have been coated with the antigen and incubate for some time. After incubation, unabsorbed antibody is washed away. Then, bovine serum albumin (BSA) is added to block any non-specific adsorption of other antibodies. Enzyme-conjugated secondary anti iso-type antibody is added and it will bind with coated antibody in the sample. After incubation the well is washed to remove all unbound and excess antibody. That substrate is added and colour produced which finally measured by spectrophotometer. Disadvantage with this technique is that immobilization is non-specific and any antibody present in the sample will attach to the plate (Abbas., et al, 2003).
Figure4: illustrates the steps of indirect ELISA. Adopted from (Jones V, 2009).
6.0 Competitive Assay
This procedure is useful to measure small molecules like heparin and many hormones. The name of the test suggests competition which will be for the available antibody sites between the antigens which are present on the sample and those coated to the plate. The antibody is first incubated with sample containing the antigen. After that complex is added to a plate coated with another antigen. Only the antibodies which are not bound to the tested antigen will bind to the coated antigen in the well. The plate is then washed and only antibody that bound to the coated antigen will remain. Then, enzyme labeled antibody is added to the plate followed by substrate which produce the colour. In this technique, the higher the concentration of antigen in sample the lower the absorbance. This test is technically more successful when using a labeled monoclonal antibody or highly specific antigens, because competition by the test sample antibody or antigen is restricted to a single target (Delves., et al, 2006).
Figure 5: shows competitive ELISA technique adapted from (Kuby, 2007).
7.0 ELISA ASSAY PRACTICAL
Some safety procedure while doing the practical:
â€¢ Gloves and lab coat should be worn during the practical.
â€¢ Used gloves should be through in provided yellow bags.
â€¢ Tips and used bloated paper must be through in a proper place.
â€¢ Special care must be taken while dealing with substrate (TMB) to avoid skin and eye contact.
â€¢ Always deal with all materials as infectious.
Materials and methods:
â€¢ Coating buffer: PBS.
â€¢ Washing buffer: 0.05% Tween 20® in PBS, pH 7.4.
â€¢ Blocking Buffer: PBS + 1% Bovine Serum Albumin (BSA).
â€¢ Diluent buffer: Phosphate Buffer Saline (PBS).
â€¢ Rabbit IgG antigen.
â€¢ Monoclonal mouse anti-rabbit IgG.
â€¢ Goat anti-rabbit IgG-HRP.
â€¢ Goat anti-mouse IgG-HRP.
â€¢ Goat anti-rabbit IgG-HRP (polyclonal antibody) .
â€¢ Monoclonal mouse anti-rabbit IgG.
â€¢ Substrate (TMB).
â€¢ Stop solution (HCL 1M).
â€¢ 96-well Microtiter plate.
â€¢ Plastic sealing paper.
â€¢ Micropipette tips.
â€¢ Unknown sample X and Y.
The aim of this practical was to detect the optimal detection and capture antibody titration, by using monoclonal mouse anti-rabbit IgG and polyclonal goat anti-rabbit IgG antibodies.
Test procedure (week 1):
100µl of coating buffer (PBS) was put in all the microtiter wells except first row (A1 to A 12).
200µl of rabbit IgG antigen added to the wells of the first row (A1 to A 12).
A serial dilution was performed for each column (from 1 to 12) by transferring 100µl of rabbit IgG antigen from the well A down to the well G and remaining 100µl from each G well was discarded.
The plate was then sealed with plastic sealing paper and incubated overnight.
It was washed and blocking buffer was used to block any unbound surface on the plate.
The plate was kept for week 2 of the practical.
Figure 6: illustrates the steps done in practical week 1
Test procedure (week2):
On week 2 of the practical, first work was done on the left half of the plate (from 1 to 6):
100µl of diluting buffer was added to the column from 2 to 6.
200µl of monoclonal mouse anti-rabbit IgG was added to wells from A to H of column1.
A serial dilution was done for each row starting by transferring 100µl of mouse anti-rabbit IgG from well number A1 up to well number A6 and the last 100µl was discarded.
Same serial dilution was done for other rows (B to H) of column 1 to 6.
The plate was then covered and incubated for 30 minutes at room temperature.
It was then washed three times (only the left side) with washing buffer and bloated in each time using bloating paper.
Figure 7: shows the steps done in practical week 2 (first part of work)
Figure 8: illustrates the steps done in practical week 2 (first part of work)
On the other half of the plate (wells from 7 to 12):
100µl of diluting buffer (PBS) was added to the column from 8 to 12.
200µl of goat anti-rabbit IgG-HRP was added to all wells of column7.
A serial dilution was done for each row starting by transferring 100µl of goat anti-rabbit IgG-HRP from well number A7 up to well number A12 and the last 100µl was discarded.
Same serial dilution was done for other rows (B to H) of column 7 to 12.
100µl of goat anti mouse IgG-HRP was added to the wells of the left half side of the plate from column 1 to column 6.
The whole plate was then covered and incubated for 30 minutes at room temperature followed by three times washing using washing buffer and bloated in each time using bloating paper.
100µl of substrate (TMB) was added to all wells.
The plate was covered to protect it from direct light contact and incubated until the required degree of blue colour was generated about 10-15min.
50µl of stop solution of (HCL IM) was added to all wells the generated blue colour changed to yellow colour.
It was then kept for reading which was done on plate reader at wavelength of 450nm.
Figure 9: illustrates the steps done in practical week 2 (second part of the practical)
Figure 10: shows the steps done in practical week 2 (second part of the practical)
Test procedure (week 3):
100µl of mouse anti rabbit IgG monoclonal antibody was added to all wells of the columns 1 and 2, wells (A3-B3) of column 3 and wells (A4-B4) of column 4.
The plate was covered with sealing paper and incubated overnight.
It was then washed with washing buffer and PBS + 1% BSA was used to block any unbound surface on the plate.
The plate was kept for week 4 of the practical.
Figure 11: shows the demonstration for plate division week 3
Test procedure (week4):
100µl of diluent buffer (PBS) was added into the wells of columns 1 and 2 except wells A1 and A2.
200µl of rabbit IgG antigen was added to wells A1 and A2.
A serial dilution for both columns 1 and 2 was done by taking 100µl from the well A1 down word to the well G1 and from A2 down ward to G2. The last 100µl from row G was discarded.
100µl of the unknown sample X was added to the wells A3 and A4.
100µl of the unknown sample Y was added to the wells B3 and B4
The plate was sealed with sealing paper and incubated at room temperature for 30 min.
It was then washed 3 times with PBS washing solution and bloated after each wash using the bloating paper.
100µl of Goat anti rabbit IgG (HRP-linked) polyclonal antibody was added only to the tested wells.
The plate was then incubated at room temperature for 30 min.
It was then washed 3 times using PBS washing solution and bloated after each wash using the bloating paper.
100µl of TMB substrate was added to the tested wells and then the plate was covered to avoid a direct light until the required degree of blue colour was generated (10-15min).
50µl of stop solution of (HCL1M) was added to the wells and the colour changed to yellow.
It was then kept for reading which was done on plate reader at the wave length of 450nm.
Figure 12: shows the steps done in practical week 4
Figure 13: shows the steps done in practical week 4
Figure 14: shows the steps done in practical week 4
Figure 15: shows the steps done in practical week 4
Week 1& 2
Mouse anti-rabbit IgG monoclonal antibody titration:
Table 1: Absorbance for the titration of mouse anti-rabbit IgG monoclonal antibody at 450 nm.
Graph 1: Different antibody concentrations for mouse anti-rabbit IgG monoclonal antibody at 450 nm.
Goat anti-Rabbit IgG HPR labeled antibody titration result
Table 2: Absorbance for the titration of goat anti-Rabbit IgG HPR labelled polyclonal antibody at 450 nm.
Graph 2: Different antibody concentrations for goat anti-Rabbit IgG HPR labelled polyclonal antibody at 450 nm.
Week 3 & 4 results
Table 3: shows the duplicated absorbance reading of standards and unknown samples ( X and Y) at 450 nm.
IgG Concentration (ng/ml)
Graph 3: shows the standard curve of Rabbit IgG, it was drown to detect the level of unknown concentration of IgG in the samples (X&Y).
Graph4: illustrate the log concentration of Rabbit IgG , it was drwn to find out the Log of unknown concentration of X&Y samples.
Calculation of the results
The equation obtained from the graph was used to calculate the unknown concentration of samples X and Y.
Y= 0.1043 x - 0.1359
Where: Y = absorbance X = concentration of unknown sample
X= 0.148+ 0.1359 / 0.1043 = 2.722
Antilog of 2.722 is 528 ng/ml
So the concentration of sample x is 528 ng/ml
Y= 0.056 + 0.1359 / 0.1043 = 1.840
Antilog of 1.840 is 69 ng/ml
So the concentration of sample y is 69 ng/ml.
This practical was divided in two parts and carried out in 4 weeks. First part (weeks 1 & 2) was aimed to find out the optimum concentration of monoclonal and polyclonal antibodies. The second part practical's weeks(3&4) was aimed to draw the standard curve by using known concentration samples to find the concentration of antibodies in unknown samples (X&Y), by using sandwich method.
The results obtained from doing titration of mouse anti-rabbit IgG monoclonal antibody are shown in table (1). The titration was done on the left side of the plate (from 1-6). That was done to find out the optimum concentration of mouse anti-rabbit IgG monoclonal antibody. The serial dilution started with the higher concentration of the antibody which is 1/2000 and the last dilution was 1/64000(lower antibody concentration). It is obvious from the graph (1) that most of the curves are linear with gradual increase in each titration. The graph (1) showed a reasonable standard curve where 1/8000 dilution gave the good optimum concentration. The 1/2000 and 1/4000 dilutions also gave a good results but they consume a lot of antibodies (excess level of antibodies) which is expensive to provide. But the 1/8000 dilution consume less antibody and it gave good results at different absorbance.
The reading in table (2) shows the results obtained from doing the titration of antibody in order to find out the optimum concentration for goat anti-rabbit IgG HPR labeled polyclonal antibody. The titration was done on the right side of the plate (from 7-12). The first dilution was 1/2000, higher concentration of antibody, and the last dilution was 1/64000 (lower antibody concentration). These results are shown in graph (2) as curves. It is obvious that the absorbance reading for all the dilutions curves flatten after the concentration of 500 ng/ml. it was found that the 1/4000 dilution is the optimum concentration for the polyclonal antibody, because it gave different absorbance reading for the antigen using less amount of antibody. So the optimum concentration for goat anti-rabbit IgG HPR labeled polyclonal antibody is 1/4000 dilution.
The second part of the practical that was done in week 3 and 4 was to prepare calibration curve in order to find out the concentration for samples X and Y. The standard calibration curve that showed in graph (3) achieved by doing duplicate titration. Also the logarithmic graph (4) was used because it is more specific and precise. After that, the average absorbance of blank was subtracted from the mean of each diluent. Then, the concentration of unknown samples (X&Y) was calculated by using the Rabbit IgG calibration curve. Finally, concentration values unknown samples (X and Y) were determined by using logarithmic graph and the equation obtained from the graph was used to calculate the concentration of unknown samples. So after calculation, the concentration of sample X was 528 ng/ml and the concentration of sample Y was 69 ng/ml.
There are many factors that can interfere with results and one of the main factors that I have noticed is the incubation time. The incubation time should be in minimum one hour whereas, were reduced to 30 minutes and that may due to insufficient reaction between antibody and target antigen on the well plate, which might be due to inaccurate absorbance reading.
The problems that can interfere with the result:
Incomplete washing of wells
Insufficient aspiration of wells
Unequal mixing of reagents
Inadequate Color Development
Conjugate or color reagent failure
Inadequate volume of substrate added to wells
Incorrect temperature or incubation time
Irregular temperature around practical area
Insufficient fixing of plate cover, leading to evaporation.
Reagents not at room temperature
ELISA is a simple, sensitive, rapid and safe technique used in many fields to measure the concentration of antigens or antibodies and required skill technique and special equipment. From my results the optimum monoclonal Mouse anti-rabbit IgG antibody concentration is 1/8000, whereas the optimum polyclonal Goat anti-rabbit IgG HRP labeled antibody concentration is 1/4000, and the concentration of unknown X is 528ng/ml and unknown Y is 69ng/ml .It is clear therefore that, ELISA technique is a reliable and accurate method.