Phosphospecific antibodies recognizing Tau
A phosphorylation, antibody technology, applied in the direction of antibodies, antibody medical components, specific peptides, etc., can solve problems such as unsatisfied
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Embodiment 1
[0503] Example 1: Production and Screening of Hybridomas and Antibodies
[0504] The purpose of this study was to generate and screen anti-Tau mAbs (monoclonal antibodies). Hybridomas were generated by fusion of spleens of Tau vaccine-immunized mice with myeloma cell lines. Hybridomas were evaluated for reactivity to phosphorylated and non-phosphorylated full-length Tau proteins and phosphorylated and non-phosphorylated Tau antigenic peptides used in vaccine preparation. Hybridoma screening was also performed by the reactivity of hybridoma supernatants to Tau tangles using immunochemistry on Tau transgenic mouse brain sections.
[0505] 1.1 Method
[0506] 1.1.1 Fusion
[0507] Wild-type C57BL / 6 mice inoculated with ACI-35 (Tau 393-408 [pS396, pS404]) were used for hybridoma production. Mice were boosted with ACI-35 vaccine on day 0 and again on day 4, and fusions were performed on day 7.
[0508] 6 × 10 from immunized mice 7 (ACI-35) spleen cells and 2×10 7 SP2-O-A...
Embodiment 2
[0527] Example 2: Cloning of Antibody Light and Heavy Chain Variable Regions
[0528] Antibody heavy and light variable region genes from hybridoma cells were cloned and the DNA sequence and location of complementarity determining regions (CDRs) and antibody binding characteristics determined.
[0529] Using Qiagen RNeasy mini kit (Cat No: 74104) from 3 × 10 6 Total RNA was prepared from hybridoma cells (1 vial). RNA was eluted in 50 uL of water and examined on a 1.2% agarose gel.
[0530] Preparation of V by IgG and kappa constant region primers using reverse transcriptase H and V K cDNA. First-strand cDNA was amplified by PCR using a larger set of signal sequence primers. Gel-purify the amplified DNA and clone into the vector T Easy (Promega). The obtained V H and V K Clones were screened for inserts of the expected size. The DNA sequence of selected clones was bidirectionally determined by automated DNA sequencing. The positions of the complementarity determi...
Embodiment 3
[0531] Example 3: Binding Study I
[0532] The objective was to measure phospho-Tau (pTau) binding of antibodies produced from subcloned hybridomas derived from mice immunized with Tau liposome vaccine. For this test, an enzyme-linked immunosorbent assay (ELISA) was used to measure the ratio of purified antibodies to phosphorylated and non-phosphorylated full-length Tau protein as well as phosphorylated and non-phosphorylated Tau antigenic peptides used in liposomal vaccine preparations. combined.
[0533] Screening was accomplished by two other methods. Immunohistochemistry (IHC) was performed on brain sections from Tau transgenic animals (TAUPIR) using an anti-Tau antibody as the primary antibody. In addition, brain protein homogenate from Tau transgenic mice was subjected to immunoblotting (WB) using an anti-Tau antibody as a blotting antibody.
[0534] 3.1 Method
[0535] 3.1.1 ELISA: Phospho-Tau Binding Assay
[0536] To test the binding of purified antibodies to...
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