1. What are the requirements for customer-provided samples when preparing alpaca single B-cell monoclonal antibodies?

Answer：

(1) Recombinant protein: ≥2.5 mg, with a protein purity >85%. The protein molecular weight should be greater than 10 kDa, and the soluble protein concentration should be between 0.5–5 mg/mL. If antigen affinity purification is required, an additional ~10 mg of recombinant protein should be provided.

(2) Peptides and small molecules: Free peptide ≥10 mg, with a peptide purity >90%, and a length of no fewer than 10 amino acids (AA). The peptide should be conjugated to KLH/BSA/OVA or another carrier protein. For small molecules, a preliminary structural assessment is required.

(3) Bacterial and viral samples: Whether they are pathogenic to humans; inactivation method; SDS-PAGE results before and after inactivation, clearly indicating sample loading amount, marker loading amount, and their respective concentrations.

2. After single B-cell screening, how are the antibodies assembled, and how are the heavy and light chain primers designed?

Answer: The heavy and light chain genes of the antibody are amplified from individual B cells and then co-expressed by transfecting cells. The primers can be designed based on the germline gene sequences.

3. What are the main components of the alpaca single B-cell monoclonal antibody preparation service?

Answer：

(1) Hapten modification/complete antigen preparation.

(2) Antigen emulsification, immunization of alpacas 4–5 times; ELISA titer determination; antiserum collection.

(3) PBMC isolation; FACS assay, ELISA assay.

(4) RT-PCR; high-throughput sequencing.

(5) Small-scale antibody transfection or scale-up production.

4. What materials are delivered as part of the alpaca single B-cell monoclonal antibody preparation service?

Answer：

(1) Modified products and their characterization results.

(2) Immune serum; ELISA test results; experimental records.

(3) High-throughput screening report, FACS binders ensuring sequence diversity; ELISA assay report, positive clones.

(4) Raw sequencing data; specific sequences.

(5) Expression plasmids, purified antibodies.

5. How to address the poor stability of monoclonal cells?

Answer：Regularly monitor the stability of monoclonal cells and select stable clones for expansion and antibody production.

6. How long should B cells be cultured before ELISA testing, and what antibody yield can be expected?

Answer：B cells typically require 10-12 days of culture before being suitable for ELISA testing, with antibody yields ranging from 0.2 μg/mL to over 10 μg/mL.

7. Will single B cell technology become the gold standard for therapeutic antibody development?

Answer：While single B cell technology is increasingly becoming one of the mainstream approaches in antibody development, there are still multiple technical routes available—such as hybridoma and phage display technologies. These methods each have their own advantages and limitations, and they maintain a complementary relationship with one another.

8. Which method yields higher PCR success rates: direct lysis of single B cells or post-culture processing?

Answer：Post-culture processing demonstrates higher PCR success rates. Cultured B cells undergo proliferation, resulting in increased cell numbers and greater template availability for PCR amplification.