Rheumatoid arthritis autoantibody conjugated antigen and applications thereof
A technology for arthritis and rheumatoids, applied in the field of bioengineering, can solve problems such as insufficient sensitivity
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[0076] Preparation method of polypeptide
[0077] The polypeptides of the present invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Correspondingly, the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
[0078]A preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology, such as Boc solid-phase method, Fmoc solid-phase method or a combination of the two methods. Solid-phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, the preferred solid phase carrier in the Fmoc system is Wang resin connected with the C-terminal amino acid in the peptide, the Wang resin structure is polystyrene, and the arm between the amin...
Embodiment 1
[0093] Example 1 Preparation, identification and storage of CCP5F-H polypeptide and its BSA coupling protein
[0094] The CCP5F, CCP5G and CCP5H polypeptides of the present invention were prepared according to conventional polypeptide synthesis methods, and their physicochemical properties were identified as follows:
[0095] The CCP5F polypeptide of the present invention is a cyclic polypeptide with a length of 18 amino acids ( Figure 4 ), a molecular weight of 2100.85 Daltons (Dal), before coupling with BSA, the amino group (-NH 2 ) end is modified as a hydrogen chain (H), and the modification at the end of a hydroxyl group (COOH) is a hydroxyl group (OH). In the Discovery HS C-18 column of HPLC, its shifting ultraviolet absorption peak at 220um wavelength is 95% A peak (main peak) in 0-2 minutes, and 5% B peak after 2-10 minutes. After the polypeptide is coupled with BSA, the concentration is still calculated according to the concentration of the CCP5F polypeptide (BSA i...
Embodiment 2
[0098] Example 2 Comparison of CCP5F, CCP5G and CCP5H polypeptide characteristics and antigen antibodies
[0099] CCP5F, CCP5G and CCP5H polypeptide BSA-coupled protein were used to immunize purebred goats in Texas, USA, respectively, in order to produce higher titer antiserum. Figure 5 To identify the reactivity (reaction titer) of the anti-CCP5F-H antibody with the antigen, the anti-CCP5F-H antiserum reacted with the corresponding antigen CCP5G polypeptide-BSA antigen protein after three-fold serial dilution, and the signal was amplified by the secondary antibody-HRP reaction Finally, different absorbance (OD) and gradient curves were shown on 96-well ELISA plates to represent the titers of goat anti-CCP5F, CCP5G and CCP5H antibodies.
[0100] The identification of goat anti-CCP5F-H antiserum ELISA titer reaction, the method and steps are: CCP5F-H is coated with 100 microliters / hole (ul / well) at a concentration of 0.5 micrograms / microliter (ug / ml) in PBS On a 96-well ELISA...
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