Sandwich assay development

Figure 8.3 Sandwich assay developed for thrombin using two different aptamers, magnetic particles, and enzymatic amplification. Two selected aptamers binding thrombin in two different, nonoverlapping sites are used. The protein captured by the first aptamer fixed onto magnetic particles is detected after addition of the second biotinylated aptamer and of streptavidin labeled with an enzyme (alkaline phosphatase). Detection of the product generated by the enzymatic reaction is achieved by differential pulse voltammetry onto screen-printed electrodes onto which the magnetic nanoparticles are deposited and kept in contact through a magnet. [From (Centi et al., 2007b).]...

SPR affinity biosensors have been developed to detect an analyte in a variety of formats. The choice of detection format for a particular application depends on the size of target analyte molecules, binding characteristics of available biomolecular recognition element, and range of concentrations of analyte to be measured. The main detection formats used in SPR biosensors include direct detection (Fig. 11), sandwich assay (Fig. 12) and inhibition assay (Fig. 13). 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Recently, two different strategies were developed for a competitive IPCR of small molecules, such as, for example, hormones, messengers, or transmitters without multiple antibody-binding sites necessary for sandwich assays. 

Two immunosensors developed by O Regan et al. [89,90] have demonstrated their usefulness for the early assessment of acute myocardial infarction (AMI). Human heart fatty-acid binding protein (H-FABP) is a biochemical marker for the early assessment of AMI. The authors constructed an amperometric immunosensor for the rapid detection of H-FABP in whole blood. The sensor is based on a one-step, direct sandwich assay in which the analyte and an alkaline phosphatase (AP) labelled antibody are simultaneously added to the immobilized primary antibody, using two distinct monoclonal mouse anti-human H-FABP antibodies. The substrate p-amino-phenyl phosphate is converted to p-aminophenol by AP, and the current generated by its subsequent oxidation at +300 mV vs. Ag/AgCl is measured. The total assay time is 50 min, and the standard curve was linear between 4 and 250 ng ml . The intra- and inter-assay coefficients of variation were below 9%. No cross-reactivity of the antibodies was found with other early cardiac markers, and endogenous substances in whole blood did not have an... 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

TBG tests that use labeled T4 have also been developed. In one commercial assay, a two-site IRMA or sandwich assay is used. TBG antibody bound to glass particles is added in excess to the patient specimen and binds virtually aU the endogenous TBG present. I-T4 is then added it binds to the TBG, forming an anti-TBG-TBG- I T4 sandwich. After a short 15-minute incubation period at room temperature, the precipitate is collected by centrifugation and its radioactivity is counted. Test values are interpreted from a calibration curve run simultaneously with the patient specimens. Unlike the typical RIA procedure, radioactivity in this IRMA method is directly proportional to the amount of TBG present in the specimen. Patients with molecular variants of TBG may have discordant results, but the assay is not affected by excess thyroxine or phenytoin. ... 

The popularity of microtiter plates in diagnostics (e.g., enzyme immunoassays) and the development of systems for washing, measurement of enzyme activity or chemiluminescence, etc., has led to scattered attempts of using these plates for hybridization. Microtiter plates are not suited for immobilization of target nucleic acid but are promising for capture and sandwich assays, particularly for large numbers of samples. 

Similarly, Coutlee et al. (1989a) developed a sandwich assay in which (i) hybrids formed in solution between the biotinylated hapten probe and the target were collected by anti-biotin antibodies immobilized in wells of a microtiter plate and (ii) detected by /3-galactosi-dase-Fab fragment (of monoclonal antibody to RNA DNA) by conversion of a substrate to a fluorescent product. Optimum conditions included hybridization for 16 h at 75°C in 2 X SSC, 10 mM HEPES, using 0.1 xg/ml probe with 7% biotinylation. 

Wide (9) and Miles and Hales (10) developed the first immunoradiometric (IRMA) assays where excess Ab was labeled. Later, two-site or sandwich assays using an excess amount of first Ab to capture the analyte from the sample matrix, and a labeled second Ab provided the signal for quantitation (11). 

This section elucidates some of the practical aspects that should be considered while developing an immunoassay. Although written with an emphasis on the sandwich assay format, and more specifically ELIS As over RIAs and ECL assays, it also applies to the competitive assay format. A systematic approach for immunoassay method development may consist of the steps shown in Fig. 3.3. 

The first SPR immunosensor for detection of pesticides was developed by Mimmni et al. [22] in the early 1990s. They used an SPR sensor developed by Biacore AB, Sweden, with the atrazine derivative bound to dextran matrix on the sensor chip. The detection of atrazine was performed using the inhibition assay and monoclonal antibodies. The sensor response was subsequently amplified by secondary antibody, which was bound to the antibody captured by the atrazine derivative (sandwich assay, see Chap. 7 in this volume [54]). This biosensor was demonstrated to measure atrazine in distilled and tap water within the range 0.05-1 ng mL in 15 min and exhibited relatively low crossreactivity with simazine and tetrabutyl atrazine (20%). The sensor surface was regenerated with 100 mM sodium hydroxide in 20% acetonitrile. 

In January 2008, Shim and Wanasundara announced their development of a sandwich ELISA that targets the Sin a 1 allergen from S. alba [11]. The assay was developed to quantify Sin a 1 levels in various yellow mustard seed lines, but was reported to be less sensitive than the previously described B.juncea assay developed by Koppleman et al. [6]. 

The assays developed (pretitrated) can all be used with competitive/inhibition systems for the detection of antibodies. Examination of antigens is more difficult since it must be ensured that the capture antibody is saturated with antigen because addition of competing antigen increases the effective concentration and free capture antibodies will bind to this. The same is applicable in indirect sandwich systems. 

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