Conformation-independent antibodies against neurotoxic tau proteins
A technology of neurotoxicity and antibodies, applied in the field of diagnosis of tau protein disease, conformation-specific antibody of phosphorylated tau protein, can solve difficult to determine and other problems
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Embodiment 1
[0055] Example 1: Preparation of typical antibodies against neurotoxic Tau protein
[0056] In this example, exemplary antibodies were prepared using phage display biopanning technology, in which a neurotoxic tau protein comprising SEQ ID NO: 8 was immobilized on a surface, and a human single-chain variable fragment (scFv) antibody library was applied to Immobilized neurotoxic tau protein to identify antibodies that bind to neurotoxic tau protein (hereinafter referred to as "binding antibody"). Finally, one or more antibodies with high affinity against the neurotoxic tau protein are selected from the bound antibodies.
[0057] First, to generate a phage library, a concentration of approximately 10 13 Phage / ml of pegylated M13K07 was used as helper phage. Reamplification of the phage library was performed and the phage library was stored in 20 aliquots at a temperature of approximately -80°C. The titer of phage stock solution in 1ml after amplification is about 8.6×10 12 . ...
Embodiment 2
[0060] Example 2: Investigating the Presence of Neurotoxic Tau in Postmortem Brains of Alzheimer's Disease
[0061] In this example, the presence and conformation of neurotoxic tau protein in post-mortem brains of Alzheimer's disease patients was assessed. First, postmortem brain tissue samples from healthy subjects and Alzheimer's patients were stained with cis and trans pT231-tau antibodies and an isotype-specific secondary antibody.
[0062] Figure 4 Immunofluorescence images showing the cis and trans conformations of phosphorylated tau protein in postmortem brains of healthy subjects and patients with Alzheimer's disease, consistent with one or more exemplary embodiments of the present disclosure . Such as Figure 4 As shown, there was no difference between the immunofluorescence (IF) intensity of healthy subjects and AD brains, and the IF intensity of both was about 3.5 million for all groups. Thus, tau phosphorylated at threonine residue 231 (pT231-tau) did not accu...
Embodiment 3
[0066] Example 3: In Vitro Evaluation of Exemplary Antibodies to Inhibit Neurodegenerative Diseases
[0067] In this example, by studying induced pluripotent stem cells (iPSCs) from healthy people, cultured Alzheimer's disease (AD) neurons and AD neurons cultured and treated with an exemplary antibody having SEQ ID NO: 1 Yuan, neurodegeneration of the three, an exemplary antibody having SEQ ID NO: 1 was evaluated in vitro. Figure 6A Immunofluorescent images of live and dead cells in healthy neurons, Alzheimer's disease (AD) neurons, and AD neurons treated with exemplary antibodies are shown, consistent with one or more exemplary embodiments of the present disclosure in the same way. Figure 6B Shown are the percentages of viable cells in cultured healthy neurons, cultured Alzheimer's disease (AD) neurons, and AD neurons cultured and treated with exemplary antibodies, with one or more exemplary antibodies of the present disclosure. The embodiment is consistent.
[0068] Suc...
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