Immunoassays microtitre plates

Murray AM, Kelly CD, Nussey SS, Johnstone AP. (1998) Production of glutathione-coated microtitre plates for capturing recombinant glutathione S-transferase fusion proteins as antigens in immunoassays. J Immunol Methods 218, 133-9. 

The use of ELISA is broad and it finds applications in many biological laboratories over the last 30 years many tests have been developed and vahdated in different domains such as clinical diagnostics, pharmaceutical research, industrial control or food and feed analytics for instance. Our work has been to redesign the standard ELISA test to fit in a microfluidic system with disposable electrochemical chips. Many applications are foreseen since the biochemical reagents are directly amenable from a conventional microtitre plate to our microfluidic system. For instance, in the last 5 years, we have reported previous works with this concept of microchannel ELISA for the detection of thromboembolic event marker (D-Dimer) [4], hormones (TSH) [18], or vitamin (folic acid) [24], It is expected that similar technical developments in the future may broaden the use of electroanalytical chemistry in the field of clinical tests as has been the case for glucose monitoring. This work also contributes to the novel analytical trend to reduce the volume and time consumption in analytical labs using lab-on-a-chip devices. Not only can an electrophoretic-driven system benefit from the miniaturisation but also affinity assays and in particularly immunoassays with electrochemical detection. 

Das Sarma, Duttagupta C, Ali E, et al. Direct microtitre plate enzyme immunoassay of folic acid without heat dena-turation of serum. /. Immunol. Methods (1995) 184 7-14. 

Enzyme immunoassays (EIA) play an important role in clinical diagnostics, veterinary medicine, environmental control, and bioprocess analysis. Antibodies are coupled to enzymes like peroxidase or phosphatase, whose products can be measured after the degradation of a substrate. Because of its high selectivity and sensitivity, EIA enables the detection of a broad spectrum of analytes in complex samples. A solid phase EIA performed in a plastic microtitre plate is called an enzyme-linked immunosorbent assay (ELISA). The coloured products produced in the ELISA can be measured spectro-photometrically rather than in a scintillation counter as for the RIA. 

A sandwich ELISA is used to search for a desired analyte in a test solution, as follows the solid phase is coated with analyte-specific capture antibodies to pull the analyte out of the test sample. After washing, the amount of analyte bound to the solid phase can be determined by adding an excess of enzyme-labelled analyte-specific antibody. The specificity of the method can be improved by using a sandwich-type, two-site assay, in which the capture and labelled antibodies have specificities for different parts of the analyte, as mentioned above. The performance of an immunoassay in a standard microtitre plate requires several hours. Such long incubation times are mostly linked to inefficient mass transport from the solution to the surface, whereas the immunocapture itself is a rapid process. 

Hanquez C, Urios P, Desfosses B, Samake H, Lince E, Rajkowski ECM, et al. A competitive microtitre plate enzyme immunoassay for plasma aldosterone using a monoclonal antibody. J Steroid Biochem 1988 31 939-45. 

Immunochemical methods for low molecular weight (<1000 D) environmental compounds, such as pesticides, nitro aromatics, and PAHs (polycyclic aromatic hydrocarbons) have been developed since more than 25 years (e.g. Hammock and Mumma, 1980 Hammock et al., 1990 Dankwardt, 2000). The most common method is the immunoassay, which is either carried out with (heterogeneous) or without (homogenous) separation steps. Heterogeneous formats that use microtitre plates, plastic tubes and/or membranes as solid supports are more commonly used. 

In most cases, competitive immunoassays for the analysis of small molecules are carried out in microtitre plates. The majority of these assays use an enzyme as label, thus leading to the term enzyme immunoassay, and the most commonly used is the ELISA (enzyme-linked immunosorbent assay) that has a heterogeneous format (separation of bound and unbound). This format can be set up either in the enzyme-tracer format (Figure 3.3.1 A) or in the coating antigen format (Figure 3.3. IB). The result shows a... 

Enzyme immunoassays are generally performed using a heterogeneous assay format on a microtitre plate containing typically 96 wells. These heterogeneous assays are referred to as enyzme-linked immunosorbent assays (ELISA). ELISAs are widely used in clinical testing. Eor example, one type of ELIS A is currently used for the clinical screening of blood supplies for HIV, the Human Immunodeficiency Virus that causes acquired immunodeficiency syndrome (AIDS). This assay is discussed in more detail below as a typical example of an ELISA. 

In the case of a non-competitive immunoassay, an excess of the specific antibody of the analyte is bound to a sohd phase, such as the wells of a microtitration plate, and the solution containing the analyte is added. After incubation and washing, a second, labeled antibody is added to the solution. After a second incubation phase the free fraction of antibody is removed and the bound fraction is quantified. 

Figure 4A outiines the basis of a simple competitive binding immunoassay where the antigen has been labelled. Antibody (preferably afifinity purified, or an IgG fraction) is coated onto a solid support (e.g. a microtitre plate well), and a constant amount of labelled antigen is allowed to compete for the antibody binding sites with vaiying amounts of unlabelled standard or sample antigen. 

For medical applications, whole cell parasites (Leishmania donovani infantum) have been used as antigens. Sol-gel encapsulation was performed directly inside the microweUs of a standard polystyrene microtitre plate currently used for immunoassays. Gelation occurs... 

Several enhancement strategies provide significant improvement of performance compared to classic immunoassays. Shortened assay times compared to standard microtitration plate-based ELISA can be achieved through miniaturisation of the working space and enhanced surface-to-volume ratio. Thus, limited diffusion... 

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