However, there is no difference between the signals produced by bound and free labelled drug so that it is necessary to separate the two before measurement. These assays are referred to as heterogeneous immunoassays. Since the amount of radio—labelled drug that remains in the solution, or is bound to the antibody, depends on the concentration of unlabelled drug, measurement of the radio—labelled drug in either the bound or the free form gives an estimate of the original concentration of unlabelled drug.
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Heterogeneous immunoassays see below using non—isotopic labels have widely replaced RIA , but in this case it is the bound fraction that is measured because a large excess of enzyme is present in the free fraction. With RIA , a calibration curve is constructed in which the percentage of labelled drug bound to the antibody is plotted against the concentration of drug.
For assays in which the drug is labelled with an enzyme or a fluorescent substance, the measurement is of an optically detected change, such as ultraviolet absorption, fluorescence or luminescence. In certain types of optical immunoassays, there is a difference between the signals generated by the free and the bound labelled—drug. Thus, no separation step is necessary and these are referred to as homogeneous immunoassays.
The difference between the signals may arise because the signal can be suppressed on binding, produced on binding or altered on binding. Both types of assay have wide application in the drug—testing field, each with its advantages and disadvantages. Detailed descriptions of different types of assay within each group are given below. Immunoassays offer a flexible approach to the analysis of various biological fluids for the presence or absence of drugs. They provide a rapid and convenient method to screen large numbers of samples in a variety of matrices and they allow the differentiation of negative specimens and thus avoid the need for further, more complicated and expensive processing or investigation.
Where specific assays are available they can be used to quantify accurately and precisely the concentration of drug in a sample as required for therapeutic drug monitoring TDM in a plasma, serum or blood sample. Drug immunoassays have been used successfully in many clinical research applications. Moore et al. More recently, Parish et al.
Immunoassay has become the method of choice for TDM and the principles and considerations described here for immunoassay are applicable to toxicology and drug screening. The main differences between these applications is the degree of quantification required TDM requires fully quantified results , and the selection of antibodies with the required cross—reactivity profiles. For example, TDM assays may require highly specific antibodies that are reactive only with a single chemical entity, whereas immunoassays used in toxicology or drug—abuse screening may be designed to be broadly cross—reactive with a whole drug group.
Labels used in immunoassay include radioisotopes, enzymes, chemiluminescent molecules, particles such as colloidal gold or latex beads and fluorescent molecules. Immunoassay formats vary from systems designed for rapid point—of care testing of a single sample through to fully automated systems that can process thousands of samples per day.
This heralded the beginning of a field of analysis that is now used routinely across the disciplines of clinical chemistry, endocrinology, pharmacology and toxicology. Antibodies raised against morphine by Spector and Parker were used in the development of an immunoassay using a radioactive tracer RIA and many variants have followed since.
However, the immunochemistry used to produce these test kits involves many intricate interactions of biochemicals with the sample under test. This chapter describes the principles of immunoassay as they specifically relate to testing for the presence of drugs and discusses the major types of immunoassay in common use. 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. Drug molecules are too small to provoke an 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 polyclonal antisera, or mice for the production of monoclonal antibodies.
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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 —COOH or —NH 2 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.
Knowledge of the metabolism of the drug and information on structurally related compounds are important when beginning the antibody synthesis. For example, Fig. 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 is produced via the 3—position it would no longer be a specific determinant against which the antibodies are raised. Hence the resultant antibody 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 cross—reactivity to different opiates varies from one antiserum or antibody to another.
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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 relative to codeine and morphine—3—glucuronide, and is expected to produce antibodies that display good cross—reactivity to 6—monoacetylmorphine 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.
This is employed as a chemical spacer between the hapten and carrier protein to allow better access to the drug for potential antibodies. 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, reversible and reaches equilibrium. High affinity antibodies bind faster than low affinity antibodies and perform better in immunochemical methods. Polyclonal antisera contain a complex mixture of antibodies raised during the immunisation process. By comparison, a monoclonal antibody is a single entity that results from the isolation of a single antibody—producing cell.
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In drug immunoassays, the higher affinity antibodies produced by polyclonal antisera can sometimes be preferable to the lower affinity antibodies produced by a monoclonal system. This produces an immune response that consists mainly of IgM followed by IgG. After analysis of the test bleeds, a larger antiserum sample can be drawn.
ELISA (Enzyme-Linked Immunosorbent Assay)
This can be as much as several hundred millilitres when using larger host animals e. Antisera yield can vary from hundreds to many thousands of tests per millilitre depending on the success of the immunisation programme and the type of assay in which they are employed.
It is not the absolute volume of serum that is important, but rather the amount and quality of antibodies contained therein. Affinity chromatography can be beneficial in certain circumstances and involves passing the antiserum over a column that contains an immobilised form of the drug of interest. In this way it is possible to fractionate a complex polyclonal antiserum.
In some cases purification is not necessary and the antiserum i. Monoclonal antibodies offer the advantage of a continuous supply of antibodies with the same characteristics, so once a good antibody is selected it can be used indefinitely. After immunisation and successful test bleeds, monoclonal antibodies are made by the fusion of mouse lymphocytes or lymphocytes from other species from the spleen with myeloma cells. The resultant hybridoma cells are separated by limiting dilution to give single cells that secrete single monoclonal antibodies.
This technique was first described in and is now in routine use Kohler and Milstein Monoclonal antibodies generally have less affinity than polyclonal equivalents, which can lead to less sensitive assays.
Monoclonal antibodies are not more specific than polyclonal antisera, but once a specific antibody is selected, the cell line can be stored and the antibody produced indefinitely. Note that in drug testing, it is possible for an antibody to be too specific, as it may be desirable to have broad cross—reactivity to a drug family such as benzodiazepines or to a single drug and its metabolites such as buprenorphine. Other molecular biology and recombinant techniques, such as phage display Chiswell and McCafferty , in which the genetic code is harnessed to produce antibodies give an exciting additional source of this important immunoassay component.
Once an immunisation is underway, the quality of the antiserum is assessed by means of an antiserum dilution curve. The resultant binding to different titres of horseradish peroxidase—labelled cotinine. BSA at 0.
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The hook or apparent peak at high antiserum concentrations is caused by a combination of steric hindrance and saturation of coating antibody to the microplate. Once the concentration of antibody titre that produces the desired response is selected, it is necessary to check that the antibody also responds as required with the target drug i.
The improved performance with subsequent test bleeds for samples taken from a rabbit immunised with a buprenorphine-protein immunogen. It is possible to gain useful information by combining both the experiments described above and analysing both a positive and negative sample at each antibody dilution. This way, binding and displacement can be seen at each antibody titre. Careful titering of the labelled drug derivative and antibody dilution can improve the assay characteristics, and the assay can be optimised further by the addition of other proteins, surfactants and stabilisers to the assay buffer.
An immunisation programme usually involves the injection of between three and six animals with the same antigen. If suitable antibodies are not produced after several immunisations, it may be necessary to start the programme again with different animals and possibly a different immunogen.