Analyte specific antibodies

A sensor array where different haptens are immobilized at well-defined areas on a plain glass surface has been developed [66], Using an automated flow injection system it was possible to incubate all areas on the chip with analytes, specific antibodies, secondary HRP-labeled antibodies, and CL substrate. Measurement of the light output via imaging performed with a CCD device allowed determination of the analytes present in the sample on the basis of the spatial localization of the CL signal. 

In the direct competitive ELISA, the analyte specific antibodies are first coated on a solid phase. The sample or standard solution of analyte is generally incubated simultaneously with the analyte enzyme conjugate or incubated separately in two steps. The amount of enzyme bound to the plate is then determined by incubation with a chromogenic substrate solution. The resulting color/fluorescence, which is inversely proportional to the analyte concentration present in the sample, is then measured instrumentally or by visual comparison with the standards. 

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 [3-galactosidase analog consists of two subunits a large dimeric polypeptide (200 kDa), denoted enzyme acceptor (EA)2, and a small polypeptide (20 kDa), denoted enzyme donor (ED). (EA)2 and ED are enzymatically inactive but spontaneously associate to give enzymatically active tetramers. In CEDIA assays, [45,46] the hapten or analyte is covalently linked to the ED, and an analyte-specific antibody is used to inhibit the assembly of the enzymatically active tetramers. Analyte in a patient s serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of active B-... 

Figure 14.6. CEDIA assay. An analyte is attached to an ED molecule in such a way that the analyte-ED conjugate recombines spontaneously with (F.A), to yield active fi-galactosidase [45]. The addition of analyte-specific antibodies inhibits spontaneous enzyme assembly [46]. Analyte in the patient s serum modulates enzyme assembly so that the signal generated by substrate turn over is directly proportional to analyte concentration.

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.

Whole serum can be used in preliminary evaluation of antibody performance, but the final lateral flow assay should be developed with purified antibody. Affinity chromatography with protein A or G can purify total antibodies from the serum, but purification on immobilized antigen is preferred, since it isolates only analyte-specific antibodies (2). Using only antibodies specific to the target helps minimize background and increase assay sensitivity. 

The protocol (Number 2) for purification of analyte-specific antibodies by affinity chromatography on immobilized antigen includes the following steps ... 

Generally, antibodies that are used as a secondary reagent are available commercially, but analyte-specific antibodies are often custom produced. The use of commercial antibodies from reputable suppliers has several advantages including the availability of a CoA, MSDS, and cross-reactivity data. Antibodies produced in-house should be similarly characterized, and the information should be documented to comply with appropriate regulatory requirements. 

Gold nanoparticles with analyte specific antibodies... 

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