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Immunoassays

In addition to their therapeutic use (Chapter 13), antibodies are frequently employed as diagnostic reagents because they exhibit extreme specificity in their recognition of a particular ligand, i.e. the antigen that stimulated their production. Antibody preparations are often used in the detection and quantification of a wide variety of specific analytes, including specific therapeutic proteins, and assays that employ antibodies in this way are termed immunoassays. The substance of interest is first employed as an antigen and injected into animals in order to elicit the production of antibodies against that particular molecule. Either monoclonal or polyclonal antibody preparations (Chapter 13) may be used in immunoassay systems.

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable 'marker'. The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay; RIA) although immunoassay systems using enzymes (enzyme immunoassays; EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags.

EIA systems take advantage of the extreme specificity and affinity with which antibodies bind antigens which stimulated their initial production, coupled to the catalytic efficiency of enzymes, which facilitates signal amplification as well as straightforward detection and quantification. In most such systems, the antibody is immobilized on the internal walls of the wells in a multi-well microtitre plate, which therefore serves as collection of reaction mini-test tubes.

Since their initial introduction over 30 years ago many variations on the basic enzyme immunoassay concept have been designed. One of the most popular EIA systems currently in use

Enzyme assay

Key:

= Immobilized ^^^ = Antigen >—= Antibody-enzyme antibody the therapeutic conjugate protein

Figure 7.B1 Basic principle of the ELISA system is that of the ELISA. The basic principle upon which the ELISA system is based is illustrated in Figure 7.B1. In this form it is also often referred to as the double antibody sandwich technique.

Antibodies raised against the antigen of interest (i.e. the therapeutic protein) are first adsorbed onto the internal walls of microtitre plate wells. The sample to be assayed is then incubated in the wells. Antigen present will bind to the immobilized antibodies. After an appropriate time, which allows antibody-antigen binding to reach equilibrium, the wells are washed.

A preparation containing a second antibody, which also recognizes the antigen, is then added. The second antibody will also bind to the retained antigen and the enzyme label is conjugated to this second antibody.

Subsequent to a further washing step, to remove any unbound antibody-enzyme conjugate, the activity of the enzyme retained is quantified by a straightforward enzyme assay. The activity recorded is proportional to the quantity of antigen present in the sample assayed. A series of standard antigen concentrations may be assayed to allow construction of a standard curve. The standard curve facilitates calculation of antigen quantities present in 'unknown' samples.

a bioassay, immunoassays are rapid (undertaken in minutes to hours), inexpensive, and straightforward to undertake.

The obvious disadvantage of immunoassays is that immunological reactivity cannot be guaranteed to correlate directly to biological activity. Relatively minor modifications of the protein product, although having a profound influence on its biological activity, may have little or no influence on its ability to bind antibody.

For such reasons, although immunoassays may provide a convenient means of tracking product during downstream processing, performing a bioassay on at the very least the final product is usually necessary to prove that potency falls within specification.

7.3.2 Determination of protein concentration

Quantification of total protein in the final product represents another standard analysis undertaken by QC. A number of different protein assays may be potentially employed (Table 7.3).

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyro-sine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyro-sine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process.

Table 7.3 Common assay methods used to quantitate proteins. The principle upon which each method is based is also listed

Method

Principle

Absorbance at 280 nm

(A280; UV method) Absorbance at 205 nm

(far-UV method) Biuret method

Lowry method

Bradford method Bicinchonic acid method Peterson method

Silver-binding method

The side chain of selected amino acids (particularly tyrosine and tryptophan)

absorbs UV at 280 nm Peptide bonds absorb UV at 190-220 nm

Binding of copper ions to peptide bond nitrogen under alkaline conditions generates a purple colour

Lowry method uses a combination of the Biuret copper-based reagent and the 'Folin-Ciocalteau' reagent, which contains phosphomolybdic-phosphotungstic acid. Reagents react with protein, yielding a blue colour that displays an absorbance maximum at 750 nm Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method

Measuring protein absorbance at lower wavelengths (205 nm) increases the sensitivity of the assay considerably. Also, as it is the peptide bonds that are absorbing at this wavelength, the assay is subject to much less variation due to the amino acid composition of the protein.

The most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye's absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also, it exhibits little quantitative variation between different proteins.

Protein determination procedures using bicinchonic acid were developed by Pierce Chemicals, who hold a patent on the product. The procedure entails the use of a copper-based reagent containing bicinchonic acid. Upon incubation with a protein sample, the copper is reduced. In the reduced state it reacts with bicinchonic acid, yielding a purple colour that absorbs maximally at 562 nm.

Silver also binds to proteins, an observation that forms the basis of an extremely sensitive method of protein detection. This technique is used extensively to detect proteins in electrophoretic gels, as discussed in the next section.

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