BIOCHEMISTRY LAB CHE554

ELISA

Approach: using the BIO-RAD ELISA Immuno Explorer Kit. (Protocol III from the BIO-RAD ELISA Immuno Explorer instruction manual, posted on the course web site.)Also see the text’s experiment 17, pages 279-282 (also see our web site).

Motivation: this is a highly sensitive, highly selective assay that is robust enough to have been distributed in kit form for use by non-scientists in the field. Thus it is one of the most commonly-used biological chemistry techniques.

Background

! ELISA: Enzyme-Linked Immunosorbent Assay.

! ELISA is a detection method, is highly adaptable, and has many uses in the ‘real world’.

! Enzyme-catalyzed colorimetric change provides rapid read-out and signal amplification.

! ELISA exploits the ability of antibodies to bind very tightly and specifically to a particular compound. The antibodies themselves are then detected by a secondary antibody linked to a visualization or quantification strategy.

! ELISA is used to diagnose diseases, identify pathogens, determine whether someone is pregnant, determine whether someone uses drugs. . . .

! This strategy can be adapted to many other uses.

Antibodies: functionality! Antibodies are proteins produced by

immune cells. ! Antibodies have high affinities

(dissociation constants ranging from 0.9 nM to as low as < 0.06 nM).

! The item recognized and bound by an antibody is called its cognate antigen.

! Some antibodies will have numerous antigens. This will occur if a variety of items all display the ‘epitope’ recognized by the antibody.

! An antigen may be recognized by several different antibodies if it displays several different epitopes.

3 4Jan 16, 2012

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Antibodies are being used as therapies to suppress rejection of organ transplants (since 1986) and to combat lymphoma and lupus and more.

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Antibodies: the molecules

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A typical antibody, or immunoglobulin, is made up of four protein molecules (it is a tetramer). There are two copies of a smaller protein, each called a “light chain” and two copies of a larger protein, each called “heavy chains”.

http://en.wikipedia.org/wiki/File:Antibody.svg

The amino termini of both the light and heavy chains are hypervariable, so that a typical human has millions of slightly different antibodies that vary with respect to amino acid sequence in that region, and thus recognition properties.

Fab: Fragment: antigen binding.

Antibodies: the molecules

7 http://en.wikipedia.org/wiki/File:Antibody.svg

Primary antibodies: to diverse antigens.Secondary antibodies: antibodies from different sources or of different types.

Atrazine

Secondary antibodies can be mass-produced more readily, and are sold as conjugates, with various enzymes that facilitate colorimetric read-out: eg. horseradish peroxidase.Hence GARHRP.

Sensitivity is 0.1 µg/L

Antibodies: structure

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http://en.wikipedia.org/wiki/File:Antibody.JPG

The light chains comprise two Ig domains, of 70-110 amino acids each, ie ≈ 7.7 - 12.2 kDa each.

Variable immunoglobulin (Ig) domains are abbreviated IgV whereas constant Ig domains are referred to as IgC. The light chain has 1 IgV and 1 IgC.

The heavy chain has 1 IgV and 3 IgC.

Light and heavy chains are held together by disulfide bonds, as are strands of the beta sheets that make up the antibodies.

http://en.wikipedia.org/wiki/File:Antibody_IgG2.png

Antibodies occur as multiples of the basic tetrameric structure, 1x, 2x and 5x. (These are referred to as monomers, dimers and pentamers). They occur in soluble as well as membrane bound form, depending on the cell type and role of the antibody.

Time course of immune response

9http://en.wikipedia.org/wiki/Immune_system

B-lymphocytes release antibodies and also bind pathogens displaying the antigen.Antibodies tag the pathogen for destruction by other components of the immune system, or impair the pathogen by inactivating molecules important for its infectivity.

T cells recognize infected human cell via antigenic peptides presented on the exterior of the infected cell as part of major histocompatibility complex formation.Lineages of B-cells and T-cells persist after the challenge and provide ‘immunological memory’.

Immune function is compromised by poor diet, alcohol, obesityHumans generate some 10 billion different antibodies

Two general strategies for the use of antibodies

Antibodies are used to detect the presence of triazine in water or human chorionic gonadotropin in urine. hCG is a peptide hormone whose levels in urine increase rapidly early in pregnancy.

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1. Antigen capture

One end of the test strip is dipped in urine an absorbent membrane wicks up urine and capillary action draws it through a reaction zone where the moving liquid also picks up enzyme-linked mouse antibodies specific to hCG (zone R, for 'reaction').

The mixture then reaches a zone where a different antibody to hCG is

anchored and there are also molecules that can be converted to a dye by the

enzyme that is attached to the first antibody (zone T for 'test'). Note that

hCG can bind to both types of antibody at once.

Fluid then moves on to a third zone where anti-mouse antibodies are anchored and more of the molecule that can be converted to dye is present (zone C, for 'control'). Anchored antibodies do not move with the fluid, and any dye formed precipitates and does not move.

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1. Antigen capture

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1. Antigen capture

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2. Antibody capture! Elisa is used to determine whether a

specific antibody is present in the serum. ! If so, this will indicate that the individual

has had prior exposure to an antigen of interest.

! If antigens characteristic of HIV are used, antibodies to HIV will be detected. Their presence can be diagnostic of HIV infection even when the virus itself if present at very low levels or hidden.

! By testing for antibodies instead of virus directly, we exploit signal amplification provided by the immune system.

! HIV infection can be detected before the virus has undergone extensive replication.

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Antibody capture

15http://www.biology.arizona.edu/immunology/activities/elisa/technique.html

Immobilized antigen is the bait.Antigen adsorbed to the polystyrene wells.

How we will do it

From the BioRad manual

Colorimetric Detection

When the detection substrate is added, it binds to the secondary antibody and is oxidized, resulting in a blue solution.

Protocol! In our experiment, we will be detecting anti-

HIV antibodies in a patients serum (this, of course, will be simulated).

! We will be conducting protocol III from the BIO-RAD ELISA Immuno Explorer instruction manual (posted on the course web site), instead of the experiment described in chapter 17 of your text.

! Make sure you read the manual and understand what is happening at the molecular level during each reaction step.

! Use the microplate reader to record your final results at a wavelength of 450 nm. Include this data in your lab report. Since your data is in triplicate, please determine the standard deviation of each of your averages. Remember to use significant figures appropriately. An average of 4.1 with a standard deviation of .2 is written 4.1 (0.2).

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Pictures (Microplate Reader)Tips for Success

! Note that the visible color should be detected 5-10 minutes after the substrate is added to the wells. The color will fade after 20-30 minutes.

! Add all solutions together very gently, otherwise bubbles will form in the wells.

! Use the microplate reader to record the absorbance of each sample at 450 nm.

! Use a new pipette tip for every reaction. Otherwise contamination could result in false positives.

! Similarly, use only every second well in the strip of tubes. The empty wells between samples provide a barrier to cross-contamination.

Changes From the Book! We will be conducting protocol III from the BIO-

RAD ELISA Immuno Explorer instruction manual (posted on the course web site), instead of the experiment described in chapter 17 of your text.

! Follow all steps (1-15) from page 77-79 of the manual. (Use 75 µl antigen instead of the 50 µl suggested.)

! Answer questions 1, 2, 4, 6 on page 80 of the manual except if ....

! For members of the class who followed protocol I instead of III (apologies from Jianing!) your questions are:1 Did your serum have ANTIGENS for the disease?

2 If you tested positive for ANTIGENS, does this mean you carry the disease? 2, 4, 6 from page 80 of the manual.

Safety Considerations! Observe all normal laboratory safety practices.! Were there any reagents in the protocol that you are

not familiar with from a safety standpoint ?