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According to the British Medical Association , an antigen is a substance that can trigger an immune response resulting in the production of an antibody as part of the body's defence against infection and disease. Most antigens are foreign proteins (not naturally found in the body), examples being microorganism or toxins. The occurrence of an antibody within the liquid portion of the blood (sera) is often as a consequence to vaccination whereby the antigen (weakened microorganism or inactivated bacterial toxins) are introduced into the body. As some antibodies are specific to various antigens they can be used outside the body in laboratory based assays to help diagnose diseases.
The ELISA technique is utilised as a serological test to determine whether or not particular antigens or antibodies are present. There are two forms of the ELISA assay. The first of which is term the direct method whereby monoclonal antibodies (mAbs) are used to test whether a particular antigen is present within a serum sample. The next assay (indirect method) entails the use of antigens in order to whether specific antibodies are present. The beneficial impact of this assay is that is can be used as a diagnostic tool for certain diseases.
In this practical, the detection of anti-viral antibodies will be done through the indirect method. The wells of the microtitre plate will be coated with four viral antigens; Measles, Mumps; Rubella and Cytomegalovirus. After which diluted sera from two patients (P1 and P2) will be added followed by washing to rid the unbound antibodies. The next stage will be the addition of a secondary anti-body (anti-human antibody) to which the enzyme is conjugated. Then finally a chromogenic substrate for the particular enzyme will be added. A colour change will be observed for by eye and the colour intensity relative to both the positive and negative control will be used to determine whether the specific antibody is present.
Microtitre plate previously coated with viral antigens
2 x 5 ml Incubation buffer (IB) for diluting patient's serum
0.5 ml 2 patient serum samples labelled P1 and P2
8.0ml Incubation buffer labelled 'conj' for diluting conjugate.
2ml Positive control serum for all 4 viruses labelled '+ve'
2ml Negative control serum for all 4 viruses labelled '-ve'
Blocking buffer (bovine serum albumin) in wash bottle (buffer)
Anti-human IgG-alkaline phosphatise conjugate (sigma)
8 ml 1mg/ ml p-nitrophenyl phosphate in glycine buffer (pH 10.4) labelled 'substrate'
5ml 3M NaOH labelled 'stop'
P200 and P20 Automatic pipettes + tips
Wad of paper towels positioned next to sink
The microtitre plate was sectioned as shown in the diagram below and the initials written using a marker pen.
2. Empty the plates over a sink and bang onto a wad of paper towels to remove excess liquid. Refill with blocking buffer only the 32 wells used in the experiment. Empty the wells as before, refill with blocking buffer, then empty for a second time before refilling all 32 wells. Leave buffer on for 3 minutes, then tip off and leave plates inverted to drain.
3. Label the two 'IB' bottles P1 and P2, dilute the patient's sera 1/100 by adding 50Âµl of the samples P1 and P2. Mix and add 200Âµl to the plate wells as shown in the diagram. There are four viral extracts and the test is carried out in duplicate.
4. Add 200Âµl of the positive control serum to the eight wells in the column three and 200Âµl negative control serum to the eight wells in column four. Incubate plate for 45 minutes at 37oC in the incubator.
5. After incubation, remove plate and rinse three times as before. Add 200Âµl of the ready diluted conjugate to all 32 wells. Incubate plates for at least 30 minutes at 37oC. Empty plates, rinse (x2) and refill wells with blocking buffer for 3 minutes.
6. Empty plate and add 200Âµl of the substrate to all 32 wells. Incubate plates at 37oC for 10 - 15 minutes to allow colour development. Read the plate 'by eye' and record which wells were positive on the plate diagram on the next page.
Below is a diagram of the assay based on that shown within The ELISA guide book.
Table 2: Definitions of symbols and terms used to describe assay
Symbols & terms
Solid phase microtitre well
Enzyme linked to reactant
Addition of reagent
1) The antigen is passively absorbed to solid phase( microtitre plate) by incubation
*Excess antigen is washed away after incubation phase.Fig. 1. INDIRECT ELISA. Antiviral antibodies react with antigen attached to solid phase (microtitre plate). Any bound antibodies are detected by the addition of anti-human antibody labelled with an enzyme. This is widely used in diagnosis.
2) Antibodies are added and incubated with solid-phase attached antigen. Those which are specific will bind to the antigen.
* Excess antibodies or non-binding component are washed away after incubation phase.
3) Antibodies labelled with enzyme (conjugate) directed against particular species in which the original antibodies were produced (anti-species). These bind to any antibodies that are attached to antigens during the incubation phase.
* Excess conjugate is washed away after incubation.
ENZ ENZ ENZ
ENZ 37 oC
4) Substrate/ chromophore is added and colour develops as a result of enzyme present.
After a period of incubation, the colour development is stopped and read by eye.
The diagram below depicts the colour changes observed after the addition of the substrate and the incubation period. As can be seen, the solution changed from a clear solution to a yellow solution when specific antibodies were present in certain wells of the microtitre plate.
Fig. 1 Microtitre plate ...
This shows that patient 1 (P1) is likely to have Mumps and Rubella as it tested positive for the presence of the specific antibodies as the clear solution changed to yellow. P1 had no observable changes in colour so this indicates that they do not have Measles or Cytomegalovirus as the specific antibodies was not present. Patients 2 (P2) tested positive for specific antibodies for Measles and Rubella as the clear solution became yellow. P2 does not have Mumps or Cytomegalovirus indicated by a lack of colour change as the specific antibodies were not present. There was also a change in the negative control which became slightly yellow; this is due to cross contamination which was probably incurred during the washing stage. As a result of this the retained results cannot be 100% trusted and it would be advisable that the experiment be repeated.
As with any experiment, there are things that can lead to the failure of the test if not executed with great accuracy and precision.
One course of action is centrifugation of the blood samples which was done beforehand. This process is required in order to precipitate the blood cells and obtain the clear liquid portion (serum). This if not done correctly will lead to cells being present which may interfere with the assay and consequentially a positive result might appear whether or not any of the four viral antibodies are present. This type of assay which may produce false positive results leads to incorrect diagnosis.
Additionally in the coating procedure of the viral antigens to the walls of the microtitre plate, if not done correctly may have led to errors. If too little antigen was added, the bare spots remaining may be adhered to by antibodies or other proteins leading to reactions that are false positive. On the contrary, if too much antigen was added then the antibodies may bind to them and are subsequently washed away leading to a false-negative reaction.
The two controls used are quite important in the experimental procedure in order to rule out errors acquired during the ELISA procedure i.e. changes in biological and chemical reagents over time and the experimental conditions. One of the controls is expected to produce a positive result if both the reagent and condition is correct whilst the other is supposed to produce a negative response. In the experiment, as can be seen on (fig), the positive control reacted as expected, although the negative control failed to react as expected. This error was possibly incurred during the washing step whereby the antibody was washed into that portion of the microtitre plate contaminating it. Because of this, the results of the patient samples cannot be trusted and hence the assay needs to be repeated.
Furthermore, the incubation step can have a profound impact on the assay. It is a way of insuring that the antigens bounded to the microtitre plate can correctly interact with the antibodies within the serum. The time allowed must be enough so that adequate binding can take place. If the time is too short then the reaction will not taken place and hence no colour will be observed at the end of the assay. Therefore the results will be incorrectly deemed to be false negatives by the observer. Temperature must be adjusted accordingly i.e. 37oC which is appropriate to the viruses otherwise denaturing might occur.
Improvement?.... Through the yielded results, it is known that specific antibodies are present although the quantity is unknown so an improvement would be to do serial dilutions of the serum samples for testing in order to determine the level of the antibodies present.
The reasons as to why antibodies are present in the serum as their formation is induce due to a vaccine supplied.
Q3. Principle of competitive ELISA used in the detection of anti-HIV antibody.