The Essay Principles And Applications Of Elisa Biology Essay

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ELA/ELISA assays were developed 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 created quantitative measurement of IgG in rabbit serum with alkaline phosphatase by using the ELISA technique. Van Weemen and Schuurs in the same year published work on EIA and concluded that one can quantify human chorionic gonadotropin concentrations in urine. They used enzyme horseradish peroxidase, coupled by means of glutaraldehyde (Lequin, 2005).

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

Principles (330)

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, more than one labelled antibody is bound to primary antibody

Flexibility, different primary detection antibodies can be used with secondary antibody

Cheap, fewer antibodies are required

Disadvantages

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

extra incubation step.

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

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 (Leinco, 2006)

Figure 3 shows that

Plate is coated with suitable capture antibody.

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

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

Detection antibody is added to the plate and binds to any antigen present in the well.

HRP is added and binds to detection antibody.

TMB substrate is added and converted to a detectable form.

Advantages

High specificity, two antibodies are used, antigen specific for captured and detected

Suitable for complex samples, antigen does not require purification for measurement

Flexibility and sensitivity,

Competitive / Inhibition ELISAhttp://www.komabiotech.co.kr/www/techniques/immunodection/images/elisaDiagrom.gif

Figure The competitive ELISA is used to quantify antigen using competitive method.

Advantages

High specificity, stwo antibodies are used, antigen is specific for captured and detected

Suitable for complex samples, antigen does not require purification before measurement

Flexibility and sensitivity

Disease detection (203)

Lyme disease

Elisa detects antibodies to B. Burgdorferi, the assay is not enough to confirm that the patient has Lyme’s disease (as the test can produce false negative results) but is distinctive enough to make the diagnosis without further testing people who live in areas infested with ticks.

Pregnancy

The most common type of pregnancy test allows the antibody to attach to a solid surface. This antibody has affinity for human chorionic gonadotropin. A combination of purified HCG coupled to an enzyme and test sample (blood, urine, etc) are added to the test. 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 added, and the amount of product is measured.

HIV

ELISA is the test used to detect infection for HIV. If antibodies to HIV are present (positive), the test is typically repeated to confirm the diagnosis. If ELISA is negative, other tests are not required. This test has a low probability 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 (173)

Food industry

The Gluten assay was designed to show low levels of gluten in food as well as in prepared and processed foods and beverages. 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 undertake changes in foods (boiled, baked, fried or heated). They discovered that hot and cold processing of foods causes milk protein to aggregate together trigger difficulties to get the milk proteins into solution, allowing them to be detected by the antibodies in ELISAs. Heating adjusts the structure of the protein, which affects 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, allowing for reliable detection of milk residue regardless of the type of processing that is used," the researchers said.

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