Essay Principles And Applications Of Elisa Biology Essay

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ELA/ELISA assays were devised separately and simultaneously by Perlmann and Engvall at Stockholm University in Sweden and by Schuurs and Van Weemen in The Netherlands (Lequin, 2005).

Engvall and Perlmann published their paper on ELISA in 1971 and established quantitative measurement of IgG in rabbit serum with alkaline phosphatase by using the ELISA technique. In the same year, Van Weemen and Schuurs published work on EIA and reported that it was possible to quantify human chorionic gonadotropin concentrations in urine. They used the enzyme horseradish peroxidase, coupled by means of glutaraldehyde (Lequin, 2005).

Figure During the early 1970s EIA/ELISA tests were on the market. Solid-phase techniques were used in the development of microtiter plates in which either an antigen or an antibody is non-covalently bound to a solid-phase (Catt, 1967). As technology progressed automated pipetting devices, multichannel pipettes, microtiter plate readers and washers were used to aid the clinician undergoing the assay; by 1980s fully automated test instruments were manufactured by Boehringer-Mannheim and Abbott. Such automated systems have come to stay in medical laboratories.

Principles (456)

Indirect ELISA is used to detect the presence of antibody.

One does this by

Adding an antigen to the substrate this causes absorption to the bottom of a well.

Blocking buffer is added to block the remaining protein binding sites.

Antibodies from a patient are then added to the coated well and allowed to bind to the antigen.

Figure Finally, enzyme-linked antibodies to human antibodies are allowed to react in the well and unbound antibodies are removed by washing.

Substrate is then applied.

Advantages

Increased sensitivity, as more than one labelled antibody is bound to a primary antibody

Flexibility, since different primary detection antibodies can be used with a single labelled secondary antibody

Cheap, since fewer labelled antibodies are required

Disadvantages

Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.

An extra incubation step is required in the procedure.

Figure Sandwich ELISAhttp://www.cellsignal.com/ddt/images/sandwich.gif

The sandwich ELISA measures the amount of antigen between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic sites capable of binding to antibody (either monoclonal or polyclonal antibodies). (Leinco, 2006)

Figure 3 shows us that

The plate is coated with a suitable capture antibody.

Blocking buffer is added to block remaining protein binding sites on plate.

Sample is added to the plate and any antigen present is bound by the capture antibody.

A suitable biotin labelled detection antibody is added to the plate and also binds to any antigen present in the well.

HRP is added and binds to the biotin labelled detection antibody.

TMB substrate is added and converted to a detectable form.

Advantages

High specificity, since two antibodies are used the antigen specifically for captured and detected

Suitable for complex samples, since the antigen does not require purification prior to measurement

Flexibility and sensitivity, since both direct and indirect detection methods can be used

Competitive / Inhibition ELISA

Figure The competitive ELISA is used to quantify antigen using competitive method.http://www.komabiotech.co.kr/www/techniques/immunodection/images/elisaDiagrom.gif

The free antigen and antibody form antigen-antibody complex.

Then the complex is bound to antigen-coated surface in the assay plate.

The unbound antibody-antigen complex is washed off before adding enzyme-linked secondary antibody against the primary antibody.

Substrate is added.

In this assay, enzyme-linked secondary antibody compete with the sample antigen which is associated with the primary antibody.

Advantages

High specificity, since two antibodies are used the antigen is specifically captured and detected

Suitable for complex samples, since the antigen does not require purification prior to measurement

Flexibility and sensitivity, since both direct and indirect detection methods can be used

Disease detection (235)

Lyme disease

Elisa is extremely useful in detecting Lyme disease process, it does this by detecting antibodies to B. Burgdorferi, the assay is not enough to confirm that the patient has e Lyme’s disease (as it can produce false negative results) but is distinctive enough to make the diagnosis without further testing people who live in areas infested with ticks that convey Lyme disease.

Pregnancy

The most basic type of pregnancy test allows the antibody to attach to a solid surface. This antibody has affinity for human chorionic gonadotropin (HCG). A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in colour of the solution.

HIV

ELISA is the first test used to detect infection for HIV. If antibodies to HIV are present (positive), the test is usually repeated to confirm the diagnosis. If ELISA is negative, other tests are not usually needed. This test has a low chance of having a false result after the first few weeks that a person is infected. (WebMD, 2010)

Lab

Figure

Figure

Absorbance

Concentration

3.301

6000

1.6395

3000

0.902

1500

0.45

750

0.26

375

0.149

187.5

0.097

93.75

0.07

46.875

0.061

23.4375

0.052

11.71875

0.045

5.859375

0.043

2.929688

Patient 1

1.643

3190

Patient 2

0.098

100

Negative control

0.07

44

Figure

Conclusion

Applications

Evidence of use of ELISA

Food industry

The Gluten ELISA assay was designed to show low levels of gluten in food ingredients as well as in prepared and processed foods and beverages. The kit is provided with a standard curve based on the levels of gluten found in cooked breads made with varying levels of wheat flour.The Gluten ELISA assay uses a monoclonal antibody (401.21) which distinguishes two important parts of the gluten, gliadin and glutenin.

Researchers studied and documented how ELISAs perform on accuracy when milk proteins undergo changes in foods (boiled, baked, fried or heated). They discovered that thermal and non-thermal processing of foods cause milk protein to aggregate together causing difficulties to get the milk proteins into solution, which enables them to be detected by the antibodies in ELISAs. These proteins also would be likely to maintain their potency once in the human body. Heating also can alter the structure of the protein, which can affect the ability of the antibody to bind to milk proteins.

“The results of these studies could be utilised by commercial ELISA kit manufacturers to aid in improving ELISAs for detection of milk residue in processed food products. These improved tests can be adopted by the food industry, if necessary, to allow for reliable detection of milk residue regardless of the type of processing that is used," the researchers said.

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