Immunodiagnostics

Topics

Definition

Presentation of various immunodiagnostic techniques

Definition

Immunodiagnostic: diagnosis based on the use of immune reactions

Immunodiagnostic: diagnosis based on the use of immune reactions

In medical diagnostics and research, immunoassays are the preferred method of determining concentration of biomolecules, e.g., hormones, cytokines, and tumor markers. Small chemical molecules also can be detected by means of these methods, so immunoassays are used in environmental and food chemistry analysis as well.

The advantage of immunoassays lies in their high sensitivity. With a sufficiently optimized test system, one can detect and even quantify in the femtomol range.

Quantitative assays The principle of many immunoassays

is based on an antigen-antibody reaction.

The antibody binds the appropriate epitope of the antigen specifically with its paratope.

This binding, which is effected by means of hydrogen bridge bonds, ion bindings, hydrophobic interactions, and van der Waals forces, is an equilibrium reaction, which is subject to the law of mass action

Quantitative assays: competitiveThe competitive assay is based on competition of two antigen populations for the same free binding sites of an antibody population in a defined volume of solution.

One of the two antigen populations consists of labeled (e.g., radionuclides, enzymes) antigens; the other is made up of similar but unlabeled antigens.

A competitive assay thus requires labeled antigens, unlabeled antigens for the standard curve, a monoclonal antibody and/or an antiserum that specifically binds to its antigen, and, of course, specimens containing antigens.

If the concentration of free binding sites is steady, and the antibody concentration, the concentration of the labeled antigens, and the fluid volumes in all starting solutions are steady as well, the quantity of unlabeled antigens in the solution can be determined.

The sigmoid course of the curve shows the reduction of the label signal in the bound fraction, with an increasing concentration of unlabelled antigens.

Quantitative assays: competitive

Radioimmunoassay (RIA) The classic RIA is a competitive assay. Therefore, an

antiserum and/or a monoclonal antibody against the antigen are required for its execution.

An unlabeled antigen and an antigen labeled with a radioisotope also are necessary.

The radioactively labeled antigen is called a tracer.

A successful RIA depends on an effective method for separating the antigen-antibody complexes from the free antigen. Only then can the radioactivity in the bound or free fractions be measured.

RIA

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A monoclonal antibody or a polyclonal antiserum can be used. This catcher antibody is coupled covalently or adsorptively to a solid phase.

It should bind the antigen in the specimen as specifically as possible. Other substances in the sample can then be removed by washing. In contrast to the competitive assay, this antibody does not work in a limiting fashion because it is applied in excess.

Quantitative assays: Sandwich

A sandwich assay requires a catcher that "fishes" the antigen from the specimen.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is the most frequently applied quantitative immunoassay today.

An enzyme is used as a label with ELISA. The antigen concentration can be determined on the basis of the substrate conversion.

ELISA A component-either the antibody or the antigen-is adsorbed to a solid phase. This allows to separate free antigen from bound antigen by a simple washing.

The sandwich and the competitive ELISA are the most frequently used; of these, the ELISA sandwich in most cases has a higher sensitivity. Also important is the direct ELISA. Here, the antigen rather than the antibody is immobilized at the solid phase. The direct ELISA is used in patient serums for the detection of antigen-specific antibodies.

ELISA in Practice The enzyme reaction is normally started by addition of the substrate

solution. This solution contains the enzyme substrate or substrates in a buffer, ensuring the pH value with which the respective enzyme works optimally.

Western blot The Western Blot enables the identification and/or

the quantification of specific proteins within a protein mixture.

Detection usually takes place by means of antibodies, which specifically bind to antigen epitopes of the target protein that is fixed on the membrane. Therefore it is also termed Immunoblot.

The detection limit for most proteins is in the pico- to nanogram range, depending on the detection system.

Western blot

A Western Blot experiment can be divided into three fundamental work steps:

Separation of a protein mixture by means of gel electrophoresis

Transfer of the proteins onto a membrane Protein detection

Western blot: Protein Detection

Membrane-bound proteins are generally detected using secondary antibodies that are labeled with radioisotopes or colloidal gold, or that are conjugated to fluorescent molecules (fluorophores) or an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

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Western blot: Protein Detection

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Western blots using fluorescent and colorimetric endpoint detection on PVDF membranes.

For all panels, 500 ng of mouse liver lysates were separated by SDS-PAGE and transferred to Millipore Immobilon-FL (fluorescent) and Immobilon-P (colorimetric) PVDF membranes.

Membranes were blocked for 1 hour with respective blockers (Panel A: 0.1x Super G in 1x PBS; B: Thermo Casein Blocker).

Blocking was followed by detection with a monoclonal anti-GAPDH antibody (1:1000) overnight in 1x PBST followed with (A) 1:10,000 anti-goat IgG-Alexa-647 and –Alexa555 or (B) colorimetric endpoint detection utilizing alkaline phosphatase with BCIP/NBT substrate (VECTASTAIN ABC System, Vector Laboratories) after a 5 minute development time. A band at 37 kDa is detectable on all blots.

A diagnostic based on diffusion of proteins on a matrix containing antibodies

Immunodiagnostics based on interaction of antibodies with cells

Blood typing

Coomb’s tests

Methods of Immune Detection. 1. Direct method: The primary antibody is

already labeled. The label could be a fluorochrome, an enzyme, or colloidal gold.

2. Indirect two step method: The species-specific, labeled secondary antibody binds to the 'naked' antigen-specific primary antibody.

3. Indirect three step method: Species-specific, labeled tertiary antibodies bind to species-specific, labeled secondary antibodies, which bind to the 'naked' antigen-specific primary antibody.

Tissue staining: Immunodetection

Detection antibodies can be labeled (conjugated, coupled) with: fluorochromes enzymes particles

Detection of fluorochromes is effected by fluorescence microscopy.

For enzymatic labels, the detection takes place by a subsequent enzyme substrate (chromogen) reaction. The soluble chromogen is immobilized at the relevant structures by the reaction, and for this a simple optical light microscope suffices for seeing and believing. An advanced level of enzymatic labels uses soluble enzyme immunocomplexes as a tertiary reagent. These are prefabricated complexes of enzyme molecules and antibodies against those enzymes.

Tissue staining: Immunodetection

Tissue staining: Immunofluorescence

Can help identify the presence of antigens in particular cells or tissues (e.g. viral antigens)

Tissue staining: Immunofluorescence

Can help identify the presence of autologous antibodies in particular tissues (e.g. in antibody-mediated diaseases)

Tissue staining: Immunohistochemistry

Can help identify the presence of antigens in particular cells or tissues (e.g. antigens that identify particular cell types)

Flow cytometry The measurement of the cells in the flow cytometer is

based on the fact that a laser beam detects the stained cells, exciting the coupled fluorescent dyes, which then emit light of a certain wavelength.

This light can be bundled and divided by a complex system of mirrors and filters in the flow cytometer.

For each different fluorescent dye one thus receives a specific signal.

In addition, independently of the coupled fluorescent dyes, a conclusion about the size of the cell and its granularity can be formed.

Flow cytometry

Flow cytometry

Cellular Assays: Cell-Mediated Cytotoxicity Chrome[51Cr]-Release Assay: This method for the

examination of cell-mediated cytotoxicity is based on the release of radioactive chrome by dead cells.

At the beginning of the test, the target cells are incubated in [51Cr]-containing culture medium for a few hours, so that the chrome isotope is absorbed into the cytoplasm.

There it binds covalently to proteins and accumulates in the cells.

Now the labeled target cells as well as the cytotoxic effector cells are put into culture for a few hours.

Cellular Assays: Cell-Mediated Cytotoxicity After a short centrifugation of the cells, the culture

supernatant is extracted and the radioactivity is quantified in a scintillation counter.

The quantity of freed 51Cr in this case is proportional to the necrotic and/or late apoptotic death of the target cells in the culture.

Different immunoassays can be used for the same purpose

SummaryDifferent immunoassays can be performed to determine:

infectioncompatibilityamounts of a particular metaboloitepresence of a particular molecule in

cells, tissues or fluidsmeasure cellular functions