Neutralizing antibodies targeting USAG-1 molecules for dental regeneration therapy
A USAG-1, antibody technology, applied in the direction of antibodies, biochemical equipment and methods, prostheses, etc., can solve the problems that tissue engineering methods have not yet reached clinical application
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Embodiment 1
[0139] Preparation of Antibody 1
[0140] To prepare mouse USAG-1 neutralizing antibody, human USAG-1 protein derived from Escherichia coli expression system (R&D systems) was used as an antigen. In a Wnt reporter gene assay using HEK293 cells, the Wnt inhibitory activity of the human USAG-1 protein derived from the E. coli expression system was confirmed ( figure 1 ). In an ALP assay using C2C12 cells supplemented with BMP7, the BMP inhibitory activity of the human USAG-1 protein derived from the E. coli expression system was confirmed ( figure 2 ). To prepare mouse USAG-1 neutralizing antibodies, three USAG-1 KO mouse lines (#116, #118, #138) were newly established using CRISPR-CAS9 ( image 3 ). Neutralizing antibodies were prepared by the iliac lymph node method using USAG-1 KO (#116) mice in ITM Co., Ltd.
[0141] 284 wells were initially screened by ELISA using the immune antigen, and a large number of positive wells were found ( Pic 4-1 and Figure 4-2 ). Ba...
Embodiment 2
[0146] Antibody In Vitro Test-1
[0147] Mouse USAG-1 (WISE) recombinant protein with PA tag added to the N-terminus was transiently expressed in Expi293F cells, and a stable expression line was established. Affinity purification was performed using the PA labeling system, and 0.2 mg PA-mUSAG-1 (WISE) was obtained from 150 mL culture supernatant ( Figure 5 ). The reduction (R) and non-reduction (NR) electrophoresis showed that the molecular weight of the purified PA-mUSAG-1 (WISE) protein was about 28kDa, which was close to the theoretical value (24kDa). The N-terminal PA-tagged mouse USAG-1 (WISE) protein derived from the Expi293F cell expression system in mammalian cells showed dose-dependent WNT signaling inhibitory activity in a WNT reporter assay ( Figure 6 ) and showed dose-dependent BMP signaling inhibitory activity in the BMP ALP assay ( Figure 7 ). The neutralizing activity of five kinds of mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, E57) w...
Embodiment 3
[0150] In vivo administration test of antibody-1
[0151] Congenital hypodontia model mice with homozygous EDA deficiency have a high loss (about 90%) of mandibular third molars (M3). A single dose of mouse anti-USAG-1 neutralizing antibody A (E37) was intraperitoneally administered to a dam carrying a model mouse for congenital tooth hypoplasia due to EDA deficiency. As a result, 7 out of 8 born EDA-deficient mice recovered the loss of the mandibular third molar (M3) ( Figure 10 ). No supernumerary teeth were observed in EDA-deficient congenital tooth hypoplasia model mice administered with mouse anti-USAG-1 neutralizing antibody A. Thus, Antibody A was found to repair missing teeth. Here, the term "restored" means that a born EDA-deficient mouse has teeth where EDA-deficient mice would normally lose teeth (without missing M3).
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