Method for differentially detecting multimeric form from monomeric form of multimer-forming polypeptides
A technology for multimer and antibody detection, which is applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., and can solve problems such as reducing the reliability of PK digestion method.
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[0070] Material
[0071] 3F4 and 3F4-biotin antibodies were purchased from Sigma (USA) and Abeam Ltd (UK), respectively. 308 and MA1-750 antibodies were purchased from Cayman Chemical Company and Affinity BioReagents Company (USA). T2-HRP and 1F5 antibodies were provided by the National Institute of Animal Health (Japan), and bovine and hamster recombinant (23-231) prion proteins were purchased from Prionics AG (CH) and Alicon AG (CH) . Mouse and human recombinant (23-231) prion proteins were provided by the University of Maryland and the National Institute of Animal Health (Japan). Age-matched normal and scrapie hamster brain homogenates were purchased from SLC Ltd. (Japan) and Baltimore Research and Educational Foundation, respectively. HRP-conjugated anti-mouse IgG and enhanced chemiluminescence kit were purchased from Amersham Biosciences (UK). PVDF membranes were purchased from Bio-Rad (USA). Startingblock was purchased from Pierce Biotechnology (USA). X-ray films w...
Embodiment I
[0072] Example 1: Western blot analysis of recombinant prion protein
[0073] Load 0.2ng of recombinant bovine prion protein (PrP 23-231, from the University of Maryland) into a non-denaturing gel without denaturing agent (12.5% SDS-PAGE) and transfer the gel contents to a PVDF membrane at 4°C (Bio-Rad) 12+ hours. PVDF blots were blocked with 50% Starting block (PIERCE) in TBS. Then, 6H4 antibody (Prionics AG) was used as primary antibody, and HRP-conjugated mouse antibody IgG (Amersham) was used as secondary antibody for detection. Blots were developed with enhanced chemiluminescence (ECL) detection by exposure to X-ray film (Fuji). Detection of prion multimers such as dimers, trimers and higher molecular bands.
[0074] As shown in Figure 3, the results show that there are multiple prion polymers and monomers in the recombinant bovine prion protein sample, and can be detected by the 6H4 anti-prion antibody.
Embodiment II
[0075] Embodiment II: for detecting PrP Sc Usability Assessment of the MDS
[0076] Plates coated with MA1-750 were prepared. Make 30 μ g MA1-750 antibody (anti-prion protein, Affinity Bioreagents company) be suspended in the 200mM MOPS of 10 μ l and enter each well of immunomodulus plate (Nunc-468667) with the volume of 100 μ l aliquots, then in 4 ℃ Incubate overnight. Plates were washed with PBS and blocked with ovalbumin in PBS for 1 hour at room temperature.
[0077] 10% bovine brain homogenate was serially diluted 4-fold in 4% zwittergent (Anaphase, USA), aliquoted into culture plates coated with MA1-750 antibody, and then incubated at 37°C for 1 hour. The MA1-750 antibody specifically recognizes the PrP c The epitope on Ser-Arg-Pro-Leu-IIe-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg, which is a non-repetitive sequence found in prions. The plate was washed 5 times with TBST and incubated with MA1-750-HRP (1:2000 in TBST) for 1 hour at 37°C. After incubation, the plates were...
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