Antibody production
All immunoassays require the use of antibodies. It is this element of the system and the way in which it is produced that is the key to the performance of the assay. Antibodies are proteins – immunoglobulins (Ig) – produced by beta-lymphocytes in response to an immunogen. There are many different immunoglobulins (IgG, IgA, IgM, IgE and IgD), but mostly IgG is used in immunoassay design. The IgG molecule can be represented simply by the letter ‘Y’ with two antigen binding frag ments, ‘Fab’ (antibody binding fragment) at the top (the arms of the Y) and the ‘Fc’ (crystalline fragment) at the base (the stem of the Y) (Fig. 1) Drug molecules are too small to pro vokean immune response, so drug immunogens are created by conjugation of a drug or drug derivative to a larger carrier protein to give an immunogen through a process called haptenisation. Host animals for antibody production are usually rabbits, sheep or goats for poly clonal antisera, or mice for the production of monoclonal antibodies. Suitable carrier proteins include bovine serum albumin (BSA), immunoglobulins or keyhole limpet haemocyanin (KLH). The aim of the haptenisation process is to conjugate multiple derivatised drug molecules (the hapten) to the carrier protein. This conjugation is usually via a 1COOH or 1NH2 moiety on the drug or via a similar group introduced by derivatisation. The position on the drug molecule used for haptenisation to the protein determines the specificity of the resultant antibodies. Know ledge of the metabolism of the drug and information on structurally related compounds are important when beginning the antibody synthesis. For example, Figure 3 shows the molecular structure of morphine, in which the carbon atoms at positions 3, 6, 2 and the N group are all readily amenable to derivatisation and thus enable conjugation to a carrier protein. The position through which the drug molecule is conjugated to the protein is ‘hidden’ from the immune system and so changes to the target molecule at the conjugation position do not usually affect the binding of the antibody. The example of morphine is a good illustration of how this can be used to the advantage of the immunoassay developer or the analyst using the assay. If an immunogen were produced via the 3-position it would no longer be a specific determinant against which the antibodies are raised. Hence the resultant anti body is likely to display cross-reactivity with the major metabolite of both morphine and diacetylmorphine (heroin), namely morphine 3-glucuronide. It would also recognise codeine (3-O-methylmorphine). The production of morphine antibodies using 3-position haptenisation is used commonly to produce broad cross-reacting ‘anti-opiate’ antibodies. The cross reactivity to different opiates varies from one antiserum or antibody to another. It is important that each antiserum or antibody be characterised fully by the assay developer.
Production of morphine antibodies via the 6 position gives better specificity to morphine rela tive to codeine and morphine-3-glucuronide, and is expected to produce antibodies that display good cross-reactivity to 6-monoacetyl morphine and the active metabolite morphine 6-glucuronide. Both of these cross-reactions may be desirable, depending on the purpose to which the antibody is being put. To produce a more specific assay for morphine, derivatisation and conjugation via the nitrogen group can be utilised. This leaves the 3- and 6 positions as the antigenic determinants and therefore produces antibodies that are more likely to be specific for morphine without cross reactivity to codeine or dihydrocodeine, for example. An N-linked antiserum will, however, give potential for cross-reactivity with molecules such as normorphine, which have an alteration at the N-position. Another consideration during antibody production is the use of a ‘bridge’ molecule. This is employed as a chemical spacer between the hapten and carrier protein to allow better access to the drug for potential antibodies. It is important to remember that the immune response in the host animal may also produce antibodies to the whole immunogen – not just the drug molecule–which in cludes the bridge molecule. Inter fer ence from ‘bridge antibodies’ can be avoided or minimised by using a different chemical spacer from that used in immunogen synthesis when conjugating the drug-signal reagent. The measure of the strength of the binding between an antigen and an antibody is described by the affinity constant. This binding is non covalent and reversible and reaches equilibrium. High-affinity antibodies bind faster than low affinity antibodies and perform better in immunochemical methods.
Figure 1 Enzyme-linked immunosorbent assay (ELISA) illustrating the principles of immunoassay. The sample is applied to the antibody which is typically coated in the wells of a 96-well plate. Labelled antigen is then added and the solution is incubated for a set period during which time the antigen (drug) and labelled antigen compete in binding to the antibody. Non-bound antigen and other non-bound substances are then washed away. The amount of bound, labelled antigen is then measured.
Figure 2 Calibration curve for an immunoassay.
Figure 3 Structure of morphine.
