Capture assays microtiter plate

Here, a sample possibly containing antigen is added to a capture system (microtiter plate wells coated with an antiserum against a specific disease). Any bound antigen is then detected by another antibody from a different species. Such assays are important m serotyping in which the second antibody... 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Another format to test for newly expressed proteins is provided through different ELISA assays. Typically, one antibody is coated on a microtiter plate and serves as a capture antibody while a second antibody (added later in the process) is labeled with a reporter molecule allowing the read-out with optical devices. These ELISAs can be operated in a quantitative manner, but need to be calibrated. The measurement unit can be traced back to the amount of protein present in the calibrator, independent of whether the calibrator consists of purified proteins or other biological materials (e.g., seeds, leaves). The amount of proteins within a plant-derived matrix (leaves, seeds, grains), however, depends on several factors, including environmental conditions and can thus not directly be related to thresholds expressed in weight-%. 

Figure 5 A penicillin-binding activity-based high-throughput SPA assay for the identification of potential inhibitors of the transpeptidase activity of PBPs. A particular PBP of interest is first labeled with biotin. The biotinylated PBP is then mixed with a 3H-labeled P-lactam in 96-well microtiter plates and the reaction mixtures are incubated at room temperature for a period of time. After incubation, the biotionylated PBP-3H-P-lactam complexes are captured by mixing with streptavidin coated SPA beads. The P-lactam-binding activity of the PBP is measured by counting the microtiter plates after mixing with the SPA beads.

Many assays that have been bead-captured and gel-based have been converted to 96-well polystyrene microtiter plates to standardize the format for automation and higher throughput. In many ways, plate formats are a bridge to the higher-density microarray formats and remain quite useful until full conversion to microarray or microfluidic formats can be made. Microtiter plate assays or activated microbeads (i.e., Luminex) for panels of cytokines are widely available because of their use in clinical medicine. 

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). 

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. 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

This assay falls into one of the four variations of an immunoassay previously defined, the antibody competition, and one of the three classes of immunoassay previously defined, the antibody capture, but without the presence of pure or partially pure antigen and without an extraneous solid support such as microtiter plates for any component of the reaction, as recommended for quantitating antigen in competition assays. 

All compounds were assayed against both herpes simplex virus-1 (HSV-1) and human parainfluenza virus type 3 (PI-3) by using Madin Darby Bovine Kidney and Vero cell lines with the aim to capture structure relationship in each of the compounds. Acyclovir and oseltamivir were used as control agents. Correlation between toxicity on uninfected cells (Vero, MDBK) and antiviral activity of the synthesis compounds were determined in the same microtiter plate. The results of the antiviral study are presented in Table 2. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

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