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 AllergensTable 45.4.The Surface Chemistries of Available Biacore ChipsChip Type Modification Type ApplicationsCM5 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 ConfigurationFor 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