One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity.

Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers. A "wild-type" scFv (scFv-wt) with a Ka value of 2.7 × 107 M-1 (at 4 °C) was prepared by linking the VH and VL domains in a mouse anti-cotinine antibody. "One-shot" random mutagenesis on the scFv-wt gene by error-prone PCR generated mutant scFv genes, which were expressed on phage particles. Repeated panning directed toward mutants with slower off-rates selected scFv clones that showed improved sensitivity in an ELISA system. One of these mutants (scFv#m1-54) with five amino acid substitutions showed more than a 40-fold enhanced Ka (1.2 × 109 M-1 at 4 °C) and, thus, was used to monitor human urinary cotinine. A limited amount of soluble scFv was reacted with urine specimens (or cotinine standards) at 4 °C for 120 min in microwells on which cotinine residues had been immobilized. The midpoint of the dose-response curves under optimized conditions (0.27 ng/assay) was more than 100-fold lower than the ELISA results obtained using scFv-wt. The limit of detection (8.4 pg/assay) corresponded to 0.17 ng/mL urinary cotinine, which was satisfactorily low for testing the threshold levels for passive smoke exposure. The assay values for volunteers correlated with the values determined using a commercial assay kit. This study evidently showed the potential of a molecular breeding approach, in which simple in vitro evolution might generate superior antibody reagents as cloned proteins, overcoming the limited molecular diversity inherent to conventional immunization-based antibodies.

[Evaluation of safe surgical treatment of peritonsillar abscess using computed tomography].

With the development of new antimicrobial agents, the incidence of peritonsillar abscess (PTA) is on the decline. PTA is still often encountered in general practice, however, where it requires immediate diagnosis and treatment. Because the internal carotid artery runs medially to the medial parapharyngeal space, damage to nearby vascular or other structures is a surgical risk of PTA. We used contrast computed tomography (CT) from PTA patients to investigate the anatomical relationship between the abscess and parapharyngeal space, and to determine safe surgical sites. We observed 31 patients with PTA--19 men and 12 women--between February 1997 and April 1999, all examined by contrast CT and undergoing drainage or incision. The average age was 30.7 years (range: 12-54 years). The abscess was on the right side in 20 cases and on the left side in 11. We determined the sites of the abscess and carotid artery, internal jugular vein, and surrounding soft tissue density area including nerves in the parapharyngeal space based on the angle and distance from recognizable anatomical structures in CT scans. The anterior margin of the parapharyngeal space was 29 +/- 5 mm posterior from the upper posterior alveolar margin. The medial margin of that space was at 15 +/- 2 degrees laterally from the midline of the incisors, and 24 +/- 4 mm laterally from the midline sagittal plane. The internal carotid artery was located medially to the parapharyngeal space, running on the sagittal plane containing the upper posterior alveolar margin. The distance from the anterior margin of the parapharyngeal space to the posterior wall of the PTA was 9 +/- 4 mm, and the distance to the anterior wall of the abscess (including the pharyngeal mucosa) was 31 +/- 5 cm. The relationship between the upper posterior alveolar margin and midline sagittal plane was useful for determining the site of the parapharyngeal space. Because the internal carotid artery is located on the same sagittal plane as the upper posterior alveolar margin, when conducting drainage or incision of PTA, we should advance sagittaLly from the point of incision to a depth of no more than 20 mm. If the tip of the instrument is kept medial to the sagittal plane of the upper posterior alveolar margin, effective treatment should be achievable without the risk of vascular damage.