A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study.

The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

Assessment of urinary pyridinoline excretion with a specific enzyme-linked immunosorbent assay in normal adults and in metabolic bone diseases.

Pyridinoline (Pyr), a specific bone resorption marker, is usually assessed in urine by high-performance liquid chromatography (HPLC) after acid hydrolysis and a prepurification step. Immunoassays have been developed to measure urinary Pyr directly. Here we developed and evaluated an enzyme-linked immunosorbent assay (ELISA), specific for the urinary free Pyr form, in normal adults and in patients with metabolic bone diseases. Urinary Pyr excretion increased significantly with age for men (r = 0.288; p < 0.001) and for women (r = 0.362; p < 0.001). An average 55% increase was noted between premenopausal (n = 41) and early postmenopausal (n = 42) women (mean +/- 1 SD; 22.4 +/- 6.3 nmol Pyr/mmol creatinine and 34.7 +/- 16.8 nmol Pyr/mmol creatinine, respectively; p < 0.001). High Pyr levels were found in patients with hyperthyroidism (n = 29; 126.5 +/- 84.2 nmol Pyr/mmol creatinine), Paget's disease of bone (n = 30; 61.8 +/- 45.8 nmol Pyr/mmol creatinine), and primary hyperparathyroidism (n = 10; 57.4 +/- 23.9 nmol Pyr/mmol creatinine). In patients with Paget's disease, urinary free Pyr excretion was correlated with urinary hydroxyproline, the conventional bone resorption marker (r = 0.87; p < 0.001), and with total alkaline phosphatase, a marker of bone formation (r = 0.55; p < 0.005). Free Pyr measured by ELISA was highly correlated with total Pyr and with total deoxypyridinoline HPLC measurements in postmenopausal women (n = 35; r = 0.94 and 0.91, respectively) and in patients with metabolic bone diseases (n = 22; r = 0.91 and 0.88, respectively; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion.

We have measured the free and peptide-bound type I collagen cross-link excretions in normal women and in patients with metabolic bone disease using the HPLC technique and immunoassays recognizing specifically the free or peptide-bound forms of pyridinoline (Pyr). After menopause, free deoxypyridinoline (free D-Pyr) excretion measured by HPLC without urine hydrolysis and expressed as a fraction of the total excretion was lower than in premenopausal women (45 +/- 15% vs. 59 +/- 12%, p < 0.005), whereas the fraction of free Pyr was not changed. In normal pre- and postmenopausal women (n = 43), the fraction of free D-Pyr was negatively correlated with bone turnover rate as assessed by the total urinary excretion of Pyr (r = -0.64, p < 0.001). In patients with a variety of metabolic bone diseases characterized by increased bone turnover (osteoporosis, Paget's disease, and hyperthyroidism), the fractions of free Pyr and free D-Pyr were significantly lower than in premenopausal controls (p < 0.001 for all diseases). After 3 days of intravenous (iv) treatment with the bisphosphonate pamidronate in patients with Paget's disease and osteoporosis, the urinary excretion of cross-linked peptides measured by high performance liquid chromatography (HPLC) or enzyme-linked immunoassay (ELISA) (NTX and CrossLaps) was markedly decreased (-52% and -85% for NTX, -71% and -93% for CrossLaps in Paget's disease and osteoporosis, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)