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Animal Immunization Platform

Animal immunization platform mainly immunizes Camelidae animals (Alpaca Liama, and Camels), laying the foundation for nanobody production.

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Animal Immunization Platform

Alpha Lifetech's animal sources are clear, with inspection and quarantine permits, clear genetic backgrounds, clear immunization frequency, and detailed records of immunization intervals, providing convenient services for antibody production. It mainly immunizes Camelidae animals (Alpaca Liama, and Camels), laying the foundation for nanobody production. In addition, it can also immunize ordinary animals (such as New Zealand white rabbits, mice, and other animals) according to the required immunization frequency and immunogenicity requirements of customers, extract and isolate them into PBMCs, and use them for subsequent experiments (such as monoclonal antibody, polyclonal antibody development, etc.). Customers can provide their own immunogens, and we will conduct strict quality control on them. If customers provide viral immunogens, they need to be inactivated and we will conduct inactivation testing to ensure safety. At the same time, we can also customize corresponding immunogens according to customer needs.

Introduction to Animal Immunization

When producing animal derived antibodies such as monoclonal antibodies, polyclonal antibodies, and nanobodies, the first step is to immunize the animals and obtain corresponding B cells, antibodies, or PBMCs to prepare for further antibody production, promote basic scientific research, develop clinical diagnostic tools, and facilitate the discovery and development of new drugs. Camels, alpacas, Liama, and other camels in Camelidae can produce nanobody (single domain antibody) libraries through immune blood collection and extraction of PBMCs. Rabbits and other animals can be immunized and antibodies can be extracted from their bodies. After purification, they can be used to produce polyclonal antibodies. Mice, rats, etc. can be used to produce monoclonal antibodies by fusing immune extracted B cells with myeloma cells. Before immunization, attention should be paid to the 3R principles (replacement, reduction, refinement), which can significantly reduce the number of animals used to the necessary minimum, improve experimental procedures, and minimize animal suffering. Proper experimental design, selection of appropriate antibodies, and strict validation procedures must also be followed.

Classification of immunogens - nanobody production

Types of Immunogens Example Immunogen Preparation Notes
Protein Immunogens Enzymes, proteins, bacterial toxins, and other substances Choose different protein expression systems: E. coli, yeast, insect cells, mammalian cells, and cell-free system Pay attention to factors such as temperature, time, pH, and inclusion bodies that affect protein expression
Nucleic Acid Immunogens DNA, RNA
DNA Immunogen: Construct plasmid DNA, clone suitable expression vectors for target genes, culture cells to amplify plasmid DNA, and extract high-purity plasmid DNA
RNA Immunogen: Need to Add Hat Structure and PolyA Tail to Prevent Degradation
When preparing DNA immunogens, attention should be paid to the appropriate host cells, and when preparing RNA immunogens, attention should be paid to preventing RNA degradation.
Viral Immunogens Whole virus inactivated vaccine, subunit vaccine, viral vector vaccine (adenovirus, lentivirus, etc.), mRAN vaccine. When preparing a whole virus inactivated vaccine, the virus is first inactivated to increase safety. Toxicity testing is used to determine if the inactivation is complete. Subunit vaccines only require virus surface proteins as antigens and need to consider immune efficacy. Adjuvants need to be added to provide immune effects, attention should be paid to the virus that needs to be inactivated, appropriate virus titers are required to ensure immune effects, and the safety of the preparation process should be strictly controlled.

Animal immunization Process or Timeline

Animal Selection And Antigen Preparation

Animal selection: Select alpacas of right age and healthy physique from healthy groups, and conduct blood examination and disease screening to ensure that there are no infectious diseases or other health problems.
Immunogen preparation: Immunogens are the key to inducing immune responses and require the selection of appropriate proteins, peptides, or other suitable substances based on the requirements of the target antibody.
A alpaca can be immunized with 1-3 antigens simultaneously, with a total antigen amount of 1-2mg per immunization and a volume of less than 2mL. Before immunization, the antigen and adjuvant are emulsified in a 1:1 ratio to form a uniform mixture, which is stored at 4 ℃.

Immunological alpaca

Record the blank alpaca ear number and start the immunization experiment. Inject into the lymph nodes near the neck of the alpaca on both sides, with 2 points on each side and approximately 0.4mL of emulsified antigen injected at each point. After immunization, observe for half an hour to confirm that the alpaca has no discomfort symptoms. Immunization should be administered every 2 weeks, with at least 4 immunizations.

Blood collection

After 4 immunizations for 5-7 days, collect 50ml of blood from the neck vein of the alpaca.

Serum separation

Blood samples are collected for immune evaluation before each antigen immunization, with 5mL of blood taken each time; On the same day, the blood was centrifuged at 400 xg for 30 minutes using a pre cooled 25 ℃ centrifuge. The upper layer of serum was separated and stored for subsequent antibody titer testing.

Separate lymphocytes

First add 15mL of cell separation solution, then slowly add 15mL of blood to a 50mL centrifuge tube. Be careful and slow when adding blood to prevent mixing of blood and separation solution. Pre cooled centrifuge at 25 ℃, centrifuge at 400 xg for 30 minutes, and store the upper serum in a new centrifuge tube at -80 ℃; Use a pipette to aspirate the middle cotton shaped upper layer immune cells into a new 50ml centrifuge tube. Add 10mL PBS buffer at room temperature to each tube, centrifuge at 25 ℃ and 400 xg for 20 minutes. Remove the supernatant, add 5mL of PBS buffer at room temperature to each tube, gently mix well, calculate the number of cells using a hemocytometer, and then centrifuge at 25 ℃ and 400 xg for 20 minutes. Remove the supernatant and dissolve the lymphocytes obtained by RNAiso Plus based on the number of cells to obtain 10 ^ 7/mL cell lysate, which is stored at -80 ℃.
antibody discovery
Fig 1:  Schematic Representation of Different Antibody Discovery Strategies and the Experimental Stages That Are Involved. (Figure source: Laustsen, Andreas H. et al.)

Customers provide immunogens-strict quality control

Types of Immunogens Requirement Quality Control Method
Peptide / Small Molecule Samples Dissolution conditions (mainly depending on whether they will inactivate phages), molecular structure/peptide sequence, synthesis report (HPLC/MS/HNMR) HPLC/MS detection
Protein Samples SDS-PAGE/WB, reconstitution conditions, buffer, label information, total amount (20-50ug), concentration, purity, etc SDS-PAGE/WB
Cell Samples Cell type (primary cells/gene edited cells), cell form (fresh/frozen thawed), growth type (adherent/suspended), culture medium requirements, number of cells provided, number of cells provided, gene edited cells to ensure stable expression of screening targets (customer corresponding primary antibody), frozen thawed cells to determine recovery rate Mainly targeting gene edited cells, using customer supplied primary antibodies for ELISA identification of target expression (our company provides HRP secondary antibodies), flow cytometry detection (our company can provide fluorescent secondary antibodies).

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  • What are the applications of animal immunity?

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    Polyclonal antibodies can be produced by immunization of animals, monoclonal antibodies can be produced by fusing B cells with myeloma cells, and PBMCs can be extracted from camel sources (alpaca, camel, alpaca, etc.) to produce various antibody fragment forms such as nanobodies, Fab fragments, scFv antibody fragments, etc.
  • What are the precautions for animal immunity?

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    To produce nanoantibodies for camel derived animals, 5-6 immunizations are required. Blood samples should be collected as a control before immunization, with an interval of 2 weeks between each immunization. After the fourth immunization, positive blood and non immunized negative blood should be collected for ELISA to determine the titer. If the titer is below 10 ^ 6, the number of immunizations should be increased until the titer reaches an appropriate level. For other animal immunizations such as rabbit immunization (the optimal or common protein antigen level for rabbit immunization is 50-1000ug), mouse immunization (the optimal or common protein antigen level for mouse immunization is 5-50ug, which requires 10 ^ 6 cells if it is a cell, and 10-50ug for nucleic acid or carbohydrates), goat immunization (the optimal or common protein antigen level for goat immunization is 250-5000ug), etc., attention should be paid to the dosage, safety, and efficacy of immunogens.
  • How to choose immune animals?

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    Depending on the type of antibody required by the customer, if you need to produce nanobodies or other antibody fragments, you can choose camel derived animals (camels, alpacas, alpacas) for immunization. If you need to produce monoclonal or polyclonal antibodies, you need to select the corresponding species based on the amount of serum, antigen, and antigen source. The maximum serum amount for rabbits is 500ml, which requires a low amount of antigen and is the primary choice for producing polyclonal antibodies. The maximum serum amount for mice is 2ml, and the maximum serum amount for rats is 20ml, which is usually the optimal choice for monoclonal antibodies. Goat produced antibodies have high affinity, and Alpha Lifetech can customize an exclusive immunization plan for you according to your requirements.
  • Do antigens need to be used together with adjuvants? How to choose?

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    There are two types of adjuvants: oil-based and water-based. Reasonable use of adjuvants is crucial for inducing strong antibody responses to soluble antigens. The sustained release of adjuvants means that fewer doses of antigens can be used, and antibody responses are more long-lasting. The first injection should be used in combination with an adjuvant. It is generally recommended to use Freund's adjuvant when the amount of immunogen is small.
  • Precautions for immunization of alpaca?

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    (1) The selection of alpaca and the antigen of immunity are the key to successful immunization. Choosing a well proportioned camel is not advisable. The purity and correct conformation of immune antigens are crucial for screening suitable antibodies for subsequent applications after immunization with alpacas. The purity of protein antigens is generally not less than 90%.
    (2) Separation of lymphocytes: Timely cell separation can effectively prevent hemolysis after blood collection to achieve the best separation effect.
    (3) The immune cycle can affect the immune response: a 1-2 week immune interval allows alpacas to have a good immune response to most antigens.
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independently developed adjuvant

Our unique formula adjuvant has been optimized for the immune system of alpaca, providing higher potency and specificity than traditional adjuvants.
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Immunization delivery
 standard

We promise to complete immunization within the specified time, provide high titer serum, and record each step through detailed reports.
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project Quality control

Each project undergoes strict quality control processes to ensure the delivery of antibodies that meet high standards to customers.
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clearly Alpaca Background

We have a healthy alpaca population that provides support for various immunization programs.
If you have any questions, please feel free to contact us at any time.

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