Food Biochemistry and Food Processing (2 edition)

BLBS102-c45 BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come

862 Part 8: Food Safety and Food Allergens

Table 45.4.The Surface Chemistries of Available Biacore Chips

Chip Type Modification Type Applications

CM5 100% carboxylation of dextran surface. General use. Routinely selected for antibody-antigen and

protein-protein interaction analysis.

CM4 30% carboxylation of dextran surface. Serum, cell extracts.

CM3 100% carboxylation of dextran surface. Serum, cell extracts.

C1 100% carboxylation of dextran surface. Permits binding events to occur closer to the sensor surface, which is

advantageous in situations where multivalent interactions occur, or

where analytes are large.

L1 Lipophilic. Lipid capturing.

SA Streptavidin surface. Detection of biotin-containing molecules. Useful for immobilisation.

NTA Nickel – nitrilotriacetic acid. Detection of histidine-tagged molecules.

HPA Flat hydrophobic surface. Used for membrane-associated interactions.

Au and SIA None. Useful for self-assembled monolayer-based interactions analysis.

coupling. This covalently captured immunoglobulin may then

be used as a bioligand to capture the pathogen from the food

sample, which is injected over the sensor surface and, therefore,

is in solution. The change in mass resulting from the biomolecu-

lar interaction introduces a change in RI, as seen by an increase

in RU, and regeneration can subsequently be used to liberate the

bacterial cell(s), so that the immobilised antibody is available

for subsequent analysis. This method of analysis can be used for

detection purposes and can be used to establish LODs. Further-

more, where required, kinetic analysis can be used to determine

the affinity of the antibody for its cognate antigen.

Assay Configuration

For food-based biosensor analysis, analytes of interest range

in size from large (intact bacterial and fungal cells) to small

(pesticide and toxin residues), requiring that the assay format

selected must be capable of providing accurate and quantitative

detection. Biacore-based assays that monitor interactions be-

tween large biomolecules, such as antibodies and proteinaceous

antigens, can be developed by immobilising either the antibody

or antigen on the surface, as the mass change introduced by the

binding of the other entity is sufficient to cause a recordable

change in RU that can be seen on a sensorgram. However, as

contaminants such as herbicides, pesticides and toxins have low

molecular weights, it is often necessary to employ an indirect

measurement method where the analyte is immobilised directly

on the sensor surface, and the larger of the binding elements

is subsequently introduced. The resultant change in mass intro-

duced by the binding of the larger element (in solution) to the

smaller, immobilised ligand can therefore be easily seen as a

change in RU. If the assay was to be performed in reverse (e.g.

the larger entity is immobilised and the small hapten or toxin is

free in solution), a small change in mass would be introduced

during an interaction event, which may be difficult to detect and

quantify reliably.

Many of the examples that are discussed in this chapter employ

antibody-based competition or inhibition assay formats (Fig.

45.3). An inhibition assay involves the combination of the sam-

ple of interest with the specific antibody before injection onto

a biosensor chip containing an immobilised target molecule.

There is competition between the immobilised and free antigen

(from the sample to be analysed) for antibody binding. A change

in signal (e.g. RU) is recorded, and this is inversely proportional

to the amount of target analyte that remains free in solution.

An alternative method to detect analytes of interest requires a

competitive assay format. In this format, the antibody, specific

to the analyte of interest, is immobilised on the surface. The

sample, containing the analyte to be determined, is mixed with a

known concentration of standard consisting of the target analyte

that has been conjugated to a large carrier protein. This results

in the analyte and the conjugated standard competing for the

immobilised antibody on the surface of the biochip. An increase

in signal is caused by the binding of the large analyte-carrier

conjugate and the data generated are similar to the inhibition

assay since the signal recorded is inversely proportional to the

amount of target analyte present in the sample. However, to per-

form these assays, it is necessary to have a suitable antibody,

and methods for antibody production are discussed in Section

‘Antibody Production Strategies’.

ANTIBODIES

Antibodies are the key biorecognition elements of the immune

system. A large variety of antibodies and antibody-derived frag-

ments have been produced with the capability of detecting an

array of structurally diverse analytes, ranging from proteins to

haptens, and these have been implemented in a number of dif-

ferent biosensor formats. Antibodies are globular glycoproteins

(sugar-containing proteins), and five main classes (or serotypes)

exist in nature, namely IgA, IgM, IgE, IgD and IgG. Single an-

tibody molecules typically have molecular weights of approx-

imately 150–200 kilodaltons (kDa). IgG antibodies (Fig. 45.4)

have a Y-shaped backbone with four polypeptide chains located

in two identical chains that are covalently attached through

disulphide bonds. The innermost chains are referred to as the

heavy chains because they are approximately double the molec-

ular weight of the outer arms (termed the light chains). The