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Principles and Program of ELISA

In this essay, I will discuss the uses of the biochemical strategy ELISA (Enzyme Linked ImmunoSorbent Assay). ELISAs are the first & most basic test used to find out whether folks are positive for certain biochemicals, such as hormones, drugs and immunoglobulins. (Glencross et al. 2010, pg 252)

Introduction

An ELISA includes immobilizing an antigen to a solid surface (e. g. a plastic multiwall dish) then obstructing the websites that continue to be unbound to prevent false excellent results. A primary antibody is then added and binds to the antigen before cleansing. A second antibody is added which is conjugated to a enzyme. A substrate is finally added which reacts with the enzyme to produce a coloured product, indicating an optimistic response (ELISA-Antibody, 2007)

The color change is assessed with a spectrophotometer which records the absorbance of the wells. The power of the color is straight proportional to the amount of antigen destined on the dish (Glencross et al. 2010 pg 398)

Various Key points of ELISA

The basis of ELISA requires immobilising antibodies onto a microtitre plate - the exact method used to prepare an ELISA plate can differ depending on the analyte being assessed. Antibodies show specificity for an analyte, allowing qualification - a test gives an optimistic or negative effect like the Avian Influenza ELISA screening process kit (Atlas Link Biotech, 2008) - or quantification - an optical thickness interpolated into a standard curve through serial dilutions as with allergen detection (McSharry et al. 2006)

There are four main "types" of ELISA - direct, indirect, sandwich and competitive.

Direct

A immediate ELISA is the simplest ELISA and quickest to perform as it uses a single most important antibody that is linked with an enzyme this complex reacts straight with the antigen which is fixed to the plate. After washing, the substrate is put into give a color change which can be measured. They could be used to check specific antibody-antigen reactions and help eliminate cross-reactivity between other antibodies. Direct ELISAs are also found in immunohistochemical staining (Thermo Fisher Scientific, 2011)a

Indirect

This format of ELISA is the most widely used in laboratories - again the antigen is fixed to the dish and there is a major antibody added. However, a second antibody-enzyme organic is added that includes a specificity for the primary antibody. The indirect ELISA is far more sensitive than direct as the primary antibodies' "signal" is amplified to give better readings. (Thermo Fisher Scientific, 2011)C:\Users\lawdunnett\Desktop\ELISA\Direct ELISA. PNG

The indirect ELISA can be used mostly for detecting the occurrence of antibodies in serum and is also trusted for discovering HIV positive patients (Richalet-Secordel and Regenmortel, 1991)

Sandwich

These ELISAs are name so due to their method - the antibody is immobilized to the microtitre dish, not the antigen. This results in the antigen being "sandwiched" between your key antibodies and the patient's antibodies (if they're present). You will discover two types of Sandwich ELISA - Two times and Triple.

Double Antibody Sandwich

This ELISA is similar to the direct ELISA as there is no indication amplification. The microtitre dish is layered with an initial antibody then the sample is packed into the plate. A secondary antibody/enzyme organic (the enzyme could be alkaline phosphatise or horseradish peroxidise) is added before finally adding a substrate to catalyse the colour change. The Increase Antibody Sandwich ELISA can be used to detect viruses in crops. (Edwards and Cooper, 1985)

Triple Antibody Sandwich

This kind of ELISA can even be called an indirect ELISA; again, the microtitre dish is covered with antibody, then antigen is added. The patient's sample is then filled prior to the antibody/enzyme organic is added.

This kind of ELISA is used to identify the patient's antibodies which may have resulted through contamination or disease (e. g. Hepatitis B, Hepatits C, HIV). For Hepatitis B, the HBV coating antibody will the wells of the plate and a artificial Hepatitis B antigen is used; the live Hepatitis B computer virus is not used as the antigen due to the danger it presents to the laboratory. The supplementary antibody/enzyme organic is usually anti-human IgG (from mice/goats etc) to interact with the patient's individuals antibody. (Wilson and Walker, 2010) Again, a substrate is added to derive a coloring change.

Competitive

ELISA Experiment

An indirect ELISA was completed on two patient samples - these samples had previously tested positive for self-nuclear antigen (ANA) and were to be tested for antibodies to extractable nuclear antigens (ENA). The ELISA was qualitative assay - patient's received a confident or negative final result in comparison with the negative control well.

The ELISA plates were layered with ENA (antigen), then anti-ENA antibodies were added. The plates were incubated a polyclonal anti-human antibody tagged with an enzyme was placed into the wells, incubated once more before adding a substrate to bring about a color change. The patient's serum was put into some of the wells rather than the anti-ENA antibodies.

The results were as below:

Discussion

The standard curve created from the ELISA shown in Body 6 is very linear and has a higher correlation - this shows a high degree of control and can give a precise positive or negative end result when the patient's serum results are compared. The error bars are somewhat large and do overlap, exhibiting there is no statistical difference between lower concentrations of Anti-ENA - there is, however a statistical difference between your higher concentrations of Anti-ENA (1500U/L and 3000U/L).

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