Method for preserving polypeptides using a sugar and polyethyleneimine
A technology of polyethyleneimine and sucrose, which is used in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0098] Antibody preparation
[0099] Suitable monoclonal antibodies can be isolated, eg, by the hybridoma method (eg, as first described by Kohler et al Nature 256:495 (1975)), recombinant DNA methods and / or subsequently from phage or other antibody libraries.
[0100] Hybridoma technology involves immunizing a host animal (such as a mouse, hamster or monkey) with a desired immunogen to stimulate lymphocytes that produce or are capable of producing antibodies that specifically bind the immunogen. Alternatively, lymphocytes can be immunized in vitro. Subsequently, the lymphocytes are fused with the myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.
[0101] As an alternative to the traditional monoclonal antibody hybridoma technique used to isolate monoclonal antibodies, antibodies or antibody fragments can also be isolated from antibody phage libraries. In particular, phage display can be used to identify antigen-binding fragme...
Embodiment 1
[0282] Example 1 - Stabilizing Calcitonin
[0283] 1. Sample Preparation
[0284] Vials of dried hCT (human calcitonin) were obtained from Sigma (Code T3535) and reconstituted in PBS to a final concentration of 3 μg / μl using the mass content indicated by the manufacturer before the experiment.
[0285] Sucrose and raffinose (sugar mixture) and PEI (Sigma catalog number: P3143-50 w / v % aqueous solution; M n 60,000) in aqueous solution. A 50 μl aliquot of vehicle was added to 3 μl hCT and the volume brought up to 60 μl with PBS. The final concentrations of sugar and PEI were:
[0286] - Sucrose: 1.03M
[0287] - Raffinose: 0.09M
[0288] -PEI: 21nM (in M n 60,000)
[0289] For controls, PBS was used instead of vehicle. Various 60 μl aliquots were prepared for testing as follows:
[0290] 1. Resuspend calcitonin in PBS and freeze
[0291] 2. Resuspend calcitonin in PBS and lyophilize
[0292] 3. Calcitonin + sugar mixture, lyophilized
[0293] 4. Calcitonin + sugar mi...
Embodiment 2
[0305] Example 2 - Preservation of Human Recombinant G-CSF
[0306] 1. Materials and methods
[0307] Material
[0308] Phospho-specific ERK1 / 2 antibodies were purchased from Sigma (Dorset, UK) and anti-ERK 2 antibodies were purchased from (Zymed UK). PEI (Mn 60,000; Sigma catalog number: P3143), sucrose (Sigma), raffinose (Fluka), PBS (Sigma), glass bottle (Adelphi glass), rubber stopper (Adelphi glass), and G-CSF (Sigma).
[0309] Sample Preparation
[0310] G-CSF lyophilized samples were reconstituted to a concentration of 10 μg / ml. 160 μl of sucrose (1.82M) and 40 μl of raffinose (0.75M) were mixed with 50 μl of PEI (150 nM concentration in terms of Mn) to complete the preservation mixture. Add 50 μl of reconstituted G-CSF solution and mix well. The final concentrations of sugar and PEI were:
[0311] - Sucrose: 0.91M
[0312] - Raffinose: 0.125M
[0313] -PEI: 25nM (in M n count)
[0314] 100 [mu]l aliquots of the final mixture were aliquoted into individual v...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More