Abstract
Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.
Highlights
Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and food samples [1,2]
Five β-lactams with high affinity to PBP2x* were selected to form complexes with PBP2x*, which were used as immunogens to immunize mice or rabbits to obtain antibodies against themselves
The results showed that there was almost no color development (OD values < 0.1, Table S1) for five PBP2x*–drug complexes, indicating that the sites of PBP2x* had been fully occupied by drugs, which could be used as qualified immunogens for subsequent immunization
Summary
Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and food samples [1,2]. The non-competitive immunoassay, generally known as the sandwich format, involves the use of two antibodies (one for capturing analyte and another labeled by an enzyme for detection), which can be added in excess [3,4]. Many attempts have been made to establish non-competitive assays for small molecules, including the use of anti-idiotypic antibodies, anti-metatype antibodies, and recombinant VH and VL to form an open sandwich immunoassay [6,7,8,9,10]. The use of anti-metatype antibodies closely resembles the sandwich immunoassay. In the present study, we screening of anti-metatype antibodies for recognizing the complexes forme and β-lactams, as well as the development of a sandwich immunoassay f.
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