Anti-thyroid hormone (T4) recombinant antibody or antigen binding fragment

A technology of thyroid hormones and binding fragments, applied in the field of antigen binding fragments and immunodiagnosis, antibodies

Pending Publication Date: 2021-01-08
TEKNOLOGIAN TUTKIMUSKESKUS VTT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This small difference between the most important thyroid hormones makes it very challenging to find specific antibodies against T3 or T4

Method used

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  • Anti-thyroid hormone (T4) recombinant antibody or antigen binding fragment
  • Anti-thyroid hormone (T4) recombinant antibody or antigen binding fragment
  • Anti-thyroid hormone (T4) recombinant antibody or antigen binding fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1. Construction of antibody gene library, selection and screening of anti-T4 antibody

[0149] T4- and T3- ​​in Freund's adjuvant The conjugates were immunized multiple times into mice. Libraries were constructed and propagated onto the surface of bacteriophage as described in Tullila and Nevanen (2017, International Journal of Molecular Sciences, 18(6), 1169). T4-AP was biotinylated with the sulfo-NHS-SS-biotin reagent following the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). Biotinylated T4-AP was incubated with a Fab fragment phage display library and captured with streptavidin-coupled high-speed beads (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic particles were washed with PBST and bound phage eluted by DTT. A total of four rounds of selection were performed before individual clone selection began.

[0150] A total of 384 single colonies were picked from the LB-amp plate and placed on a 96-well microtiter plate con...

Embodiment 2

[0152] Example 2. Affinity and specificity determination

[0153] The affinity of purified anti-T4 Fab fragments for various analytes was analyzed using a surface plasmon resonance instrument BIAcore T200 (GE Healthcare, Piscataway, NJ, USA) with an inhibition assay (Nieba, L. et al. 1996, Anal. . Biochem. 234, 155–165; Badescu, G.O. et al. 2016, PLoS ONE, 11, e0152148; Persson, B.D. et al. 2009, J. Virol. 83, 673–686; Tullila, A., and Nevanen, T.K. 2017, International Journal of Molecular Sciences, 18(6), 1169; Adamczyk, M. et al. 1998, Bioconjugate chemistry, 9(1), 23-32. Before immobilization, as Cannon et al. (2004, Analytical biochemistry, 330.1:98-113) The surface of a BIAcore CM5 sensor chip (29104988, GE Healthcare, Piscataway, NJ, USA) was pretreated as described. T4-AP and AP (P5931, Sigma-Aldrich, St.Louis, MO, USA) were dissolved in 10 mM sodium acetate pH 4.0 (BR100351, GE Healthcare, Piscataway, NJ, USA) was diluted to a concentration of 100 μg / mL. T4-AP and AP ...

Embodiment 3

[0157] Example 3.ELISA assay

[0158] L-thyroxine

[0159] Coat microtiter plate wells (Nunc, 436023) with 100 μl of 4 μg / ml T4-BSA (Fitzgerald, 80-IT50) in PBS (15 mM phosphate, 150 mM NaCl, pH 7.3) at +37°C with shaking at 600 rpm )One hour. Wells were then washed three times with 300 μl PBS supplemented with 0.05% (v / v) Tween 20 (Sigma-Aldrich, 93773-250G). Spray the wells with 200 μl of (Thermo Fisher Scientific, 37516) was blocked for 30 minutes and washed as above. 100 μl of the pre-incubated mixture containing 0.25 nM anti-T4 Fab and different concentration of L-thyroxine (T4) (Sigma-Aldrich, T2376), transferred to the wells and incubated at room temperature for 30 minutes with shaking at 600 rpm. Each sample has 12 replicates. Wells were washed as above, and 100 μl of goat anti-mouse κ-HRP (SoutherBiotech, 1050-05) diluted 1:1000 in Superblock was transferred to the wells and incubated for 30 minutes at room temperature with shaking at 600 rpm. The wells were w...

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Abstract

The present invention relates to the fields of antibodies, antigen binding fragments and immunodiagnostics. Specifically, the invention relates to a recombinant anti- body or antigen binding fragmentfor binding thyroid hormone T4 (thyroxine) and halogenated bisphenol A. Also, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the recombinant antibody or antigen binding fragment of the present invention, as well as an expression vector and host cell comprising the nucleic acid molecule of the present invention. Still, the present invention relates to a method of producing a recombinant antibody or antigen binding fragment for binding T4 thyroid hormone and halogenated bisphenol A, a test kit and an immunoassay comprising the recombinant antibody or antigen binding fragment of the present invention, and a method for determining T4 thyroid hormone and / or halogenated bisphenol A levels in a sample of a subject. Still further, the present invention relates to a method of treating a sample, e.g. an immunoaffinity-based sample preparation method for enrichment of the halogenated bisphenol A from a sample.

Description

[0001] field of invention [0002] The present invention relates to the fields of antibodies, antigen-binding fragments and immunodiagnostics. In particular, the present invention relates to recombinant antibodies or antigen-binding fragments for binding the thyroid hormone T4 (thyroxine) and halogenated bisphenol A. Also, the present invention relates to isolated nucleic acid molecules comprising a nucleotide sequence encoding a recombinant antibody or antigen-binding fragment of the present invention, as well as expression vectors and host cells comprising the nucleic acid molecules of the present invention. Furthermore, the present invention relates to methods for producing recombinant antibodies or antigen-binding fragments for binding T4 thyroid hormone and halogenated bisphenol A, test kits and immunoassays comprising recombinant antibodies or antigen-binding fragments of the present invention, and methods for determining Method for T4 Thyroid Hormone and / or Halogenated B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26A61P25/00
CPCC07K16/26C07K2317/55C07K2317/92A61P25/00
Inventor 塔贾·内瓦宁亨利·阿罗拉安蒂·图利拉朱哈·鲁维宁塔贾·帕克基宁皮耶兰杰洛·梅特兰戈洛
Owner TEKNOLOGIAN TUTKIMUSKESKUS VTT
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