Analyte-specific capture antibody

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

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. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

The competitive assay is another format used to quantitate an analyte. An unlabeled analyte competes with a labeled analyte (enzyme-conjugated molecule) for binding to a specific capture antibody (Figure 11.1c). 

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. 

In order to detect the analyte specifically, a complex has to be formed first. To this end, the revelation moiety (e.g. an enzyme-labelled antigen or antibody) is for instance incubated in the chip so as to bind to the analyte that has previously been captured within the microchannel. In another scheme, the analyte solution is first mixed with the revelation moiety, and the formed complex is then incubated in the chip in order to be captured on the bed of antibodies coating the walls of the micro-channel. After a washing step (to remove the excess affinity partner), the microchannel is filled with the substrate which shall thus react... 

Noncompetitive (also known as two-site, sandwich, labeled antibody, or immunometric methods) (see Chapter 9) require two antibodies capable of simultaneously binding PTH (Figure 49-14) (1) a capture antibody immobilized to a solid phase, and (2) a signal or reporter antibody labeled with a measurable substance or an enzyme changing the concentration (substrate or product) of a measurable substance. Unlike competitive immunoassays, with noncompetitive methods, both antibodies are added in excess, ensuring that aU analyte is measured. After formation of the ternary complex or sandwich, excess labeled antibody is removed by washing before quantification of complexes. Noncompetitive immunoassays provide increased sensitivity, specificity, reproducibility, and convenience. 

Generally, polyclonal antibodies are easier to produce, and high-affinity polyclonal antibodies can be obtained. Monoclonal antibodies are more specific to a certain epitope. They provide continuous production of exactly the same defined reagent and are more preferable for excess-reagent assays. The double sandwich technique has used two antibodies from monoclonals or combinations of mono- and polyclonals, with specificity against two different epitopes of the analyte. One antibody functions as a capturing antibody for the analyte and the other as the label carrier (118). 

Fig. 3. Competitive Immunoassay (A) In the absence of analyte, analyte-conjugated liposomes are captured by an analyte-specific antibody at the competition zone. (B) In the presence of analyte, analyte-tagged liposomes compete with analyte present in the sample for the antibody immobilized at the competition zone yielding a signal that is inversely proportional to the analyte concentration.

Larger analytes are readily detected by direct interaction with antibodies immobilized on the sensor surface (Fig. 9.5.6). Sensitivities for this type of assay are typically in the high nanomolar to high picomolar range. The sensitivity and specificity can be increased simultaneously with the use of secondary antibodies that are specific for an epitope other than the one bound by the surface immobilized antibodies. If very high sensitivity is required, several stages of amplification can be carried out, allowing for detection of femtomolar levels of protein. For example, mouse monoclonal capture antibodies can be immobilized on the sensor surface. 

Viruses, whole cells and spores can also be detected by direct interaction with antibodies immobilized on the sensor surface (Fig. 9.5.7). If a genus specific antibody is used as the capture antibody, species-specific antibodies can be used to speciate the captured analyte. 

---