Cleavage and ligation reactions can be assayed by monitoring either the appearance of reaction products or the disappearance of substrates. For example, protein or peptide proteolysis can be monitored by following the appearance of cleavage products. Cleavage products may be separated from substrate by gel electrophoresis or by chromatographic separation and monitored by UV absorption or colorimetric assay. Similarly, polynucleotide ligation can be monitored by following the appearance of ligation products.
If the cleavage or ligation activity is low, the detection signal may require amplification to detect the reaction products. For example, radio labeled substrates may be synthesized and the resulting reaction products may be detected by radio immunoassay. Alternatively, if the substrate is conjugated to an enzyme which catalyzes a colorimetric reaction, the resultant reaction products may be detected by means of an enzyme immunoassay. Highly sensitive enzyme immunoassays have been developed. When employed at their limit of sensitivity, the signal produced by an enzyme immunoassay in response to the presence of reaction product becomes comparable to the background signal.
In the catalytic antibody field, antibody libraries are routinely screened in order to identify catalytically active antibody. There are two limitations to creating a useful catalytic antibody, viz.: 1.) designing a productive immunogen which is an analog of the substrate or reaction intermediate and producing an antibody library therewith; and 2.) screening the resultant antibody library for the desired catalytic activity. In short, the limitations are display and detection. The problem of display can be facilitated by converting the antibody diversity into a combinatorial library in phage where the recognition and replication functions are linked in a single entity and monitored by simple binding events. However, the screening of phage particles which display only a small number of catalytic antibody molecules requires a highly sensitive assay methodology. In such instances, the displayed catalytic activity may be only slightly higher than background activity. Prior art methods for assaying cleavage and ligation reactions sometimes lack the requisite sensitivity for identifying small quantities of low activity antibody. In some instances, catalytic antibody having a low level of catalytic activity can be useful if it is the only antibody identified to have such catalytic activity and/or if it is employed in an "evolutionary scheme" for generating antibodies having higher levels of catalytic activity. Accordingly, the sensitivity of the assay employed for screening an antibody library, may be the limiting factor with respect to the identification of useful antibody. The ease or difficulty of the assay may also limit the willingness of workers to perform these assays.
In instances in which one can obtain antibodies to the reaction product or substrate, an immuno-PCR assay may be constructed and employed as a detection system. (T. Sano et al., Science (1992): vol. 258, pages 120-122). An immuno-PCR assay is similar to an enzyme immunoassay except that the enzyme-antibody conjugate is replaced by an antibody conjugated to a PCR amplifiable polynucleotide strand. Immuno-PCR assays are highly sensitive. However, at very low levels of antigen, the immuno-PCR assay is limited by non-specific binding of the antibody-polynucleotide conjugate.
What is needed is a highly sensitive assay for detecting cleavage or ligation reactions. The assay should not employ an antibody conjugate and should have the lowest possible background signal. The assay should be labor efficient and should be adaptable for assaying any cleavage or ligation reaction.