1、What information is needed for antibody development?
Answer: Preliminary information includes the following points:
(1) Research species: Specific types, such as animals, plants, microorganisms.
(2) Target antigen information: Whether it is a small molecule, protein, antibody, and whether there is sequence information. Technical analysis is conducted based on the provided sequence.
(3) Antibody application: WB, IHC, IF, Elisa antibody pairs, Flow, IP. Different applications have different workloads in the later stages, leading to different quotations.
(4) Quotation: The final quotation reference is determined based on the difficulty of application analysis.
2、Briefly describe the advantages of your company's platform?
Answer: Currently, our antibody development platform utilizes the latest single B-cell recombinant rabbit monoclonal antibody development platform. It has high throughput, providing 20-100 clone supernatants at once. Antibody affinity is relatively 10-100 times higher than that of murine sources. The final delivery includes antibody gene sequences and recombinant expressed antibodies.
3、How many sequences are delivered for antibodies?
Answer: Generally, 1-3 distinct sequences are delivered. If more diverse clone sequences are needed, charges are applied based on the quantity gradient.
4、How much recombinant antibody is delivered in the end?
Answer: The final delivery is 1mg of recombinant antibody. If more is needed, it can be scaled up for production based on the standards of 1L production.
5、How long does it take from antigen development to antibody development?
Answer: Antigen preparation generally takes 5-6 weeks, immunization 8-9 weeks, B-cell screening 3-4 weeks, molecular amplification vector construction 3 weeks, and recombinant expression 2 weeks. The total timeline is usually 4.5 months.
6、How is antibody potency determined?
Answer: For rabbit immunization, after the fourth immunization, we select rabbits with serum potency reaching a dilution ratio of 1:128K for subsequent B-cell screening. If the rabbit's potency is insufficient, an additional 1-2 boosters will be administered. The supernatant from subsequent B-cell culture will be provided to researchers for screening and validation for their own applications. The best clones are then selected for molecular cloning and recombinant expression, and the final antibody potency generally meets the requirements for the researcher's intended applications.
7、How many rabbits are immunized for a project? Are B cells sourced from spleen cells or PBMCs? At what standard is B-cell enrichment screening performed?
Answer: Currently, routine projects immunize two rabbits. If the antigen's immunogenicity is good enough to ensure sufficient positive B cells for later screening, we include both spleen cells and PBMCs in the initial positive B-cell screening. Generally, after the fourth immunization, if the rabbit's serum potency reaches a dilution ratio of 1:128K, B-cell enrichment screening is initiated.
8、In the ELISA screening stage of B-cell supernatants, are 5 plates used? What is the approximate volume per sample?
Answer: Yes, for projects with low demand for candidate clones, we generally use 5 plates, which are sufficient to obtain positive clones. After Elisa screening and identification of each well, 100-150μl of the candidate clone can be provided to the client for additional verification.
9、What is the approximate positivity rate during the ELISA and neutralization screening stages for projects currently being run by Mabnus?
Answer: The positivity rate varies significantly depending on the project type. Generally, the initial screening positivity rate for 5 plates of Elisa is between 15% and 60%, and the subsequent neutralization screening in the Elisa candidate clones ranges from 3% to 55%.
10、In the screening stage, different projects may have different screening methods. If there are multiple detection items, does Mabnus handle them independently, or can both parties collaborate to complete the work?
Answer: We prefer collaborative testing and screening to ensure the success rate of candidate molecules on the customer's application platform and to improve identification efficiency.
11、The document mentions a case where only one sequence was delivered in the end. What is the usual delivery standard for a project?
Answer: For a normal project, we generally deliver 1-3 unique positive clone sequences.
12、Does Manbus have experience in antibody discovery for peptide targets, including immunization with KLH-coupled antigens?
Answer: Currently, we have experience in developing both peptide and small molecule projects, including antigen synthesis, design, coupling, etc. Usually, the candidate clone repetition rate is higher in such projects, and the final delivery of clones is not too much.
13、How is the charge determined if the number of screenings is increased?
Answer: We need to evaluate the antigen situation first. If an increase in the number of screenings is needed, we provide a quotation based on a gradient standard according to the required quantity.
14、How does Mabnus determine the standard for positive clones? Does it require an OD value greater than a certain threshold or an S/N ratio greater than a certain value?
Answer: The positive determination is based on an OD value more than 5 times that of the negative control.
15、For peptide projects, does the repetition rate mean that B-cell diversity is weak during screening? Will the positivity rate be lower than other forms of antigens?
Answer: Because the epitopes of peptides are limited, antibodies recognizing these peptides may have relatively consistent sequences, leading to less diversity than protein antigens. There won't be a significant difference in the positivity rate.
16、Is the project delivery based on the delivery of B-cell positive supernatants?
Answer: During the screening process, we provide some B-cell positive supernatant to customers for testing and screening. After confirming the positive clones, we continue to analyze, clone, and recombinantly express the clones. After further validation, qualified antibodies are delivered, providing 1-3 confirmed antibody sequences and recombinant antibodies. If an increase in the delivery quantity is required, a gradient quotation is provided based on the demand.
17、Can B-cell culture supernatants from the project be directly used for blocking assays?
Answer: For routine Elisa blocking verification experiments, the supernatant from a single well of B-cell culture is sufficient for 1-2 rounds of testing.