There are a lot of variations of immunoassays which are available to be used in laboratories. However, few of them which are commercially available, are convenient to use for illicit/abused drug testing programs which are run at various workplaces. Depending on market demand, immunoassays can be automated, with little preparation, inexpensive and give results quickly without consuming much time. Some of the commonly used testing systems are as follows:
RIA - Radioimmunoassay
RIA was introduced by Yalow and Berson, based on the principle of using radioactive labels such as isotopes of iodine (I125, I131) or tritium (3H). Radioummunoassays are also known as heterogeneous as the parts of the assay should be physically separated before measuring the reaction products. Competitive RIA is dependent on the reaction between antigen and antibody where the labeled analytes compete with donor analytes to bind to the site present on the antibody which is there in the reagent or the surface. After being incubated for some time, the bound part of the analyte is separated from the free part by either decantation or precipitation. A scintillation counter checks for the radioactivity of the bound segment. These counts are inversely proportional to the quantity of drug in the sample which is known by the interpolation of a curve. RIA is a very sensitive test which generates strong signals which are easily detected. Even though, RIA was commonly used for earlier workplace drug testing programs, it is now replaced by many non-isotopic homogenous systems that are faster, easily automated and do not have issues regarding radioactive waste disposal.
EIA – Enzyme Immunoassay
EIA also known as Enzyme Immunoassay involves the use of enzymes rather than radioactive isotopes to detect relevant compounds in a sample. These tests utilize the catalytic action of enzymes and are now commercially used assay systems. Enzyme labels, along with relevant substrates are used to generate fluorescent, colored or luminous end products which can be optically measured. Single enzyme molecules cause many other molecules of substrate per unit to be converted, thus enhancing the sensitivity and strength of the assay as compared to labels producing a single event. However, the sensitivity of such assays is limited by the range of photometric detectors which are used to measure the quantity of reaction products.
The labels on the enzymes can generate several kinds of signals, which depend on the substrate they are present in. The easiest way to measure common drug testing systems is via spectrometer which measures the colorimetric signal generated by the enzymes. Enzyme labels such as G6DP – Glucose-6-phosphate dehydrogenase, horseradish peroxide and β-galactosidase are quite commonly used.
An EIA functions via enzymes which are coupled to drug or antigen in the reagent. The antigen/enzyme particles compete with the drug in order to bind with the antibody. If no drug is present in the sample, the antigen/enzyme would bind to the antibody and the activity of the enzyme would be limited. If the drug is present in the sample, it would bind to the antibody, and the free enzyme available in the sample reacts with the substrate and forms some byproducts via catalyzation. EMIT – Enzyme Multiplied Immunoassay Technique is one such example of a reagent created by Syva Company in 1970. This test depends on the change in absorbance at 340nm due to reduction of NAD to NADH due to the action of the enzyme G6PD. This competitive, homogenous system is based on the concentration of the drug present in the sample being directly proportional with the magnitude of change in absorbance. A scheme of EMIT is described diagrammatically in Figure 2.3. Currently, some vendors give similar assay systems.
CEDIA – also known as Cloned Enzyme Donor Immunoassay is homogenous, competitive EIA which has somewhat different approach to it. This assay depends on genetically formulated enzyme β- galactosidase having two units: EA – Enzyme Acceptor unit and ED – Enzyme Donor unit. Even though, these units are inactive, they activate and reassemble in the solution so that they can react with the substrate. The ED analyte of the enzyme competes with specimen analyte for the antibody binding site, the bound ED part doesn’t combine with EA unit to activate an enzyme. If a drug is found in the donor’s sample, the unbound ED part reassembles and reacts with the substrate and causes a change in the color absorbance (See Figure 2.4). This change is basically proportional to the quantity of drug found in the donor sample.
Both types of EIA are competitive, homogenous assays which can be automated and are commonly utilized as many laboratory analyzers. The main use of EIAs is the initial testing in the drug testing program in US currently.
Another variant of EIA, known as ELISA – Enzyme Linked Immunoabsorbent Assays are known as heterogeneous assays as both, the unbound and bound parts of the reaction are separated before the results are declared. Traditional ELISA has one component immobilized or absorbed to the solid surface, such as a micro titer plate. In this assay type, the antibody is stationary, and addition of the sample and enzyme labeled drug conjugates is done to the well where they compete for sites on the stationary antibody to be bound to them. Washing is done to remove the unbound material and then, a chromogenic material is added to form colored by product with the bounded enzyme. A spectrometer reads the results according to the absorbance being inversely proportional to the concentration of drug found in the sample.
Compared with EIA, ELISA has enhanced sensitivity and specificity as far as the technological constraints are concerned. But, the illicit drugs in the urine sample are present above the sensitivity of EIA tests, so that’s not a problem as such. ELISA however, is not easy to automate and consumes more time to test as compared to EIAs as the incubation time is longer. Therefore, ELISA is not preferable for routine illicit-drug testing. However, when more specific analysis is needed, then ELISA is the preferred choice. The are many laboratories though, which use ELISA to test illicit-drugs in urine samples.
Fluorescence enabled labels are used to increase the sensitivity of immunoassays. Even though, fluorescence enabled product can be used for enzyme assays, the basic use of fluoroimmunoassays is to directly use fluorophore labeling in the FIPA – Fluorescence Polarization Immunoassay. The FPIA is competitive assay which is homogenous where the drug/antigen of the fluorescein labeled reagent and the test specimen compete for sites to bind on the antibody. Polarized plane light is used to excite the mixture and the rate of polarization of fluorescence is judged by the rate of rotation of the fluorescence-antigen conjugate when it is in its excited state in the sample solution. Small unbound conjugate revolves quickly and has lower polarization as compared to larger bound conjugates that revolve slowly and are more polarized. Therefore, the degree of polarization of the sample is inversely proportional to the concentration of the analyte. (See Figure 2.5)
Kinetic Micro Particle Immunoassay
KIMS – also known as Kinetic Interaction of Micro particles in solution, is a technology that immunoassays utilize to conduct agglutination assays. In this method, the particulates such as cells, latex particles, etc clump together which indicate that a primary reaction has taken place between the antibody and the antigen. Some of the registered brands that use this type of assay methodology in the urine drug testing are: Roche Diagnostics and the Abu screen. It is shown in Figure 2.6, that the first generation assays are founded on the competition which occurs between a labeled drug imitative which is conjugated with a latex particle and the test sample’s drug in order to bind to the antibody. This binding of the particles to the antibody results in clumping/aggregate formation, which increases the absorbing capacity of the mixture. If the test sample’s drug binds to the antibody, then clumping is inhibited which decreases the absorbance. Therefore, the absorbing capacity of the sample is inversely proportional to the concentration of the drug. The assays shown in Figure 2.6 illustrate that the micro particles get bounded with the antibody rather than drug conjugate to improve assay’s performance.
Lateral Flow Immunoassay
In the later years of 1970s, point-of-care testing appliances started being commonly utilized for workplace drug testing regimes. A lot of these appliances function on competitive, membrane-dependant assay formats which can detect many compounds in one-step only. The basic benefit of these appliances if that lay personnel can perform the tests at the point of collection and forward only the non-negative tests to the laboratory for further confirmation. This saves both time and expenditure of specimen transportation.
The lateral flow testing system reacts the antigen with the antibody while removing excess reactants by chromatogenic separation. This process is shown in Figure 2.7 below. The reagent of the detector (antibody coated with labels such as gold, etc) is dried on the sample where it interacts with the added sample as it goes through the membrane of the testing strip. Near the capture line, the detector reagents react with the capture reagent which is halted on the membrane and forms a line. A test line and a control line shows the result visually which is then further evaluated. If a drug is positively present in the sample, the antibodies which are labeled are bound to it and therefore, it is not available for reaction with capture reagent. Therefore, line development shows negative results, where as an absence of line shows non-negative result. However, a drawback of this technique is the visible confirmation of the presence of line, which is subjective to the operator’s visual capacity. Therefore, more techniques have evolved to evaluate the result properly.