Generation of a Novel Monoclonal Antibody against Cortisol-[C-4]-Bovine Serum Albumin Conjugate: Application to Enzyme-Linked Immunosorbent Assay for Urinary and Serum Cortisol

Kobayashi, N.; Sun, Peng; Fujimaki, Yayoi; Niwa, Toshimitsu; Nishio, Takehiko; Goto, Junichi; Hosoda, Hiroshi

doi:10.2116/analsci.18.1309

Cited by 23 publications

(14 citation statements)

References 27 publications

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“…Cortisol is used as a biomarker for the functions of the hypothalamic–pituitary–adrenal axis 25 and thus practical anti-cortisol antibodies have always been in great demand as diagnostic reagents. However, only a few publications have demonstrated the production of monoclonal antibodies capable of targeting serum or urinary cortisol 26 – 28 . Immunoassay sensitivities closely correlate with affinities of the antibodies used 10 .…”

Section: Resultsmentioning

confidence: 99%

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 10 5 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved K a values (> 10 10 M −1 ), compared with the wild-type scFv ( K a = 3.8 × 10 8 M −1 ), none of which could be recovered via conventional panning procedures operated for the entire library.

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Section: Resultsmentioning

confidence: 99%

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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“…The specificity of antibodies against haptens is significantly influenced by the position on the hapten molecule used for conjugation with a carrier protein to yield an immunogen. 29 The measured affinity constant for the Mab-Cortisol 3 used in the rotating structure assay is approximately 2.1 × 10 7 M -1 . (USBiological, catalog no.…”

Section: S(t)mentioning

confidence: 99%

“…Monoclonal antibodies (Mab) are highly desired for high selectivity and assay performance standardization. 29 However, Mab for haptens typically have lower affinity constants, and washing steps can greatly diminish the signal by decreasing the amount of antigen and/or labeled antigen. The specificity of antibodies against haptens is significantly influenced by the position on the hapten molecule used for conjugation with a carrier protein to yield an immunogen.…”

Section: S(t)mentioning

confidence: 99%

Detection of FITC-cortisol via Modulated Supraparticle Lighthouses

et al. 2006

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Hormones are important bioactive compounds in blood and tissue that vary in concentration in response to stress and certain disease states. Establishing the changes in physiological hormone concentrations over time can lead to more effective diagnoses and perhaps a better understanding of the evolution of stress and disease. To monitor concentration over time, the sampling must be rapid and noninvasive; specimens such as saliva that require little effort to collect are preferred. However, more sensitive assay techniques are needed when compared to blood analysis since free hormone concentration in saliva is only a small fraction of the concentration in circulating blood. In this work, magnetic field-induced structures of paramagnetic particles are used as a solid substrate to demonstrate improved detection limits for a separation-free assay of cortisol. Once formed, the structures are subjected to a rotating magnetic field and this leads to two important features. First is the ability to utilize frequency and phase filtering (lock-in amplification) for the signal generated from surface-bound labeled species. Second is the improved mass transport of the antigen to the surface of the rotating structures. These two unique capabilities result in a quantifiable signal at a relatively low target antigen concentration. This method has been demonstrated with the detection of fluorescein isothiocyanate-labeled cortisol (FITC-cortisol) at a concentration of 300 pM.

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“…Now, monoclonal antibodies are attractive, because the supply of a characterised antibody can be sustained (82, 83, 84). Some changes in hapten have assisted antibody quality (85). A few methods are based around anti-idiotype assays (86, 87).…”

Section: Immunoassays Of Steroidsmentioning

confidence: 99%

Steroid Assays in Paediatric Endocrinology

Honour¹

2010

JCRPE

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Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.Conflict of interest:None declared.

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Generation of a Novel Monoclonal Antibody against Cortisol-[C-4]-Bovine Serum Albumin Conjugate: Application to Enzyme-Linked Immunosorbent Assay for Urinary and Serum Cortisol

Cited by 23 publications

References 27 publications

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Detection of FITC-cortisol via Modulated Supraparticle Lighthouses

Steroid Assays in Paediatric Endocrinology

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