Hybridoma & Vaccines Flashcards

Question

What are the six main strategy points to be followed for the production of a monoclonal antibody (mAb)? Mention and elaborate on them.

Answer 1

Immunization Immortalisation HAT selection Detection of prod. Ab Cloning production of Mab 1. Immunization Goal: To elicit a strong and specific immune response in the host animal. Antigen Selection: Choose an antigen relevant to the intended application. The antigen should be well-characterized and capable of eliciting a robust immune response. Immunization Protocol: Administer the antigen to the host animal (commonly mice) along with an adjuvant to enhance the immune response. Multiple injections (boosts) are often given over several weeks to ensure a high titer of antibodies. Monitoring Response: Collect blood samples periodically to measure antibody titers and ensure that the immune response is developing as expected. 2. Immortalization Goal: To create hybrid cells (hybridomas) that can produce antibodies indefinitely. Harvesting B Cells: After a strong immune response is confirmed, spleen cells (rich in antibody-producing B cells) are harvested from the immunized animal. Cell Fusion: Fuse the harvested B cells with myeloma cells using a fusion agent like polyethylene glycol (PEG). This process combines the antibody-producing capability of B cells with the immortality of myeloma cells. Hybridoma Formation: The fused cells (hybridomas) can grow indefinitely and produce the specific antibody of interest. 3. HAT Selection Goal: To select hybridoma cells that have successfully fused and are capable of producing antibodies. Selective Medium: Hybridomas are cultured in HAT (Hypoxanthine-Aminopterin-Thymidine) medium. This medium selects for successfully fused hybridomas by exploiting the fact that unfused myeloma cells cannot survive in HAT medium, while normal spleen cells have a limited lifespan. Screening for Viability: Only hybridomas (fused cells) can survive and proliferate in HAT medium, allowing for the selection of successful fusions. 4. Detection of Productive Antibody (Screening) Goal: To identify hybridoma clones that produce the desired antibody. Primary Screening: Use techniques like ELISA, Western blotting, or immunofluorescence to screen the culture supernatants from hybridoma wells for the presence of antibodies specific to the target antigen. Secondary Screening: Further characterize positive clones for specificity, affinity, and functionality to ensure they produce high-quality antibodies. 5. Cloning Goal: To ensure monoclonality of the antibody-producing cells. Limiting Dilution: Perform limiting dilution or other cloning methods to isolate single hybridoma cells. This step ensures that each colony originates from a single cell, resulting in monoclonal antibodies. Verification: Confirm the monoclonality of the clones by additional rounds of screening and testing to ensure that the antibodies produced are homogeneous and specific. 6. Production of Monoclonal Antibodies (mAb) Goal: To produce and purify monoclonal antibodies at a larger scale. Expansion: Once stable hybridoma clones are identified, they are expanded in culture to produce larger quantities of the antibody. Purification: Purify the antibodies from the culture supernatant using affinity chromatography (e.g., protein A/G columns), ensuring high purity and concentration. Quality Control: Conduct rigorous quality control tests to verify the purity, concentration, and functionality of the monoclonal antibodies. This includes testing for contaminants, aggregation, and consistent performance in relevant assays

Answer 2

1. Limited Lifespan of B Cells Finite Lifespan: Primary B cells have a limited lifespan in culture and cannot proliferate indefinitely. This makes it impractical to use them for long-term production of antibodies. Senescence: Even if B cells could be cultured for a short period, they would eventually undergo senescence and die, ceasing antibody production. 2. Insufficient Yield Low Antibody Production: Primary B cells produce antibodies, but the yield is typically low and not sufficient for large-scale production required for therapeutic, diagnostic, or research applications. 3. Lack of Immortality No Continuous Cell Division: B cells do not have the ability to divide indefinitely. To produce monoclonal antibodies continuously, a cell line with the capability for unlimited division (immortalization) is required. 4. Variability and Instability Genetic Instability: B cells can be genetically unstable and may lose their antibody-producing capability over time. Variability in Antibody Production: Different B cells produce different antibodies, and without a way to isolate and continuously propagate a single antibody-producing cell, it is impossible to maintain consistency

Answer 3

1. Selection in HAT Medium HAT Medium Composition: HAT medium contains hypoxanthine, aminopterin, and thymidine. Hypoxanthine: A precursor for the salvage pathway of nucleotide synthesis. Aminopterin: A folic acid antagonist that blocks the de novo synthesis of purines and pyrimidines. Thymidine: A precursor for DNA synthesis. Role of HGPRT: HGPRT is an enzyme involved in the salvage pathway of purine synthesis. It allows cells to utilize hypoxanthine to produce nucleotides when the de novo pathway is blocked by aminopterin. 2. Selective Growth of Hybridomas Myeloma Cells Without HGPRT: Myeloma cells are chosen to lack HGPRT to ensure they cannot use the salvage pathway to synthesize nucleotides in the presence of aminopterin. Without this pathway, they cannot survive in HAT medium. B Cells With HGPRT: Normal B cells, which have functional HGPRT, can use the salvage pathway to survive in HAT medium, but they have a limited lifespan and will eventually die. Hybridoma Survival: Fusion of B Cells and Myeloma Cells: When B cells (which have HGPRT) are fused with myeloma cells (lacking HGPRT), the resulting hybridoma cells inherit the HGPRT enzyme from the B cells. Selective Growth: Only successfully fused hybridomas, which have both immortality from myeloma cells and the HGPRT enzyme from B cells, can survive in HAT medium. Unfused myeloma cells die because they cannot use the salvage pathway, and unfused B cells die due to their limited lifespan. 3. Ensuring Monoclonality and Specificity Elimination of Unfused Cells: The use of HGPRT-deficient myeloma cells ensures that only hybridomas survive in HAT medium, eliminating unfused myeloma cells and any other non-hybrid cells. Hybridoma Stability: Hybridomas are stable and capable of indefinite proliferation, continuously producing the specific monoclonal antibody of interes

Answer 4

A vaccine is a biological product that can be used to safely induce an immune response that confers protection against infection and/or disease on subsequent exposure to a pathogen.

Answer 5

The vaccine must contain antigens that are either derived from the pathogen or produced synthetically to represent components of the pathogen

Answer 6

To protect us, through engagement of adaptive immunity, against disease or conditions that possess a threat to our health.

Answer 7

Vaccines stimulate a protective immune response. After a mild infection there will be protective immunity so that immunological memory remembers the pathogen if we get re-infected.

Answer 8

Vaccination is the most effective mean of controlling infectious diseases

Answer 9

Initiation of an immune response against a certain pathogen by injection of a vaccine, a dead or attenuated (non-pathogenic) form of the pathogen or something else that may induce immunity against it.

Answer 10

Transfer of an active component (e.g. antiserum or purified antibodies) that protects against disease in the short term, as long as the transferred antibodies remain active in the body.

Answer 11

Short term--> Antibody response (B cells) T helper cells Cytotoxic T cells Herd immunity Prevention of infection vs disease possibly vaccines prevent infection and the development of disease after infection with a pathogen (difficult to distinguish) Non-specific effects: immunization with some vaccines perturbs the immune system in such a way that there are general changes in immune responsiveness that can increase protection against unrelated pathogens. Immune memory Long-term

Answer 12

1) Vaccine injected into muscle 2) Protein Ag taken up by DCs (activated through pattern recognition receptors by danger signals in the adjuvant) 3) Trafficked to draining lymph nodes 4) Peptide presentation- MHC -> T cell activation + signalling (by soluble Ag) through B cell receptor ➔ T cells drive the B cell development in the LN ➔ Maturation of the Ab response increases Ab affinity and induces different Ab isotypes ➔ Production of short-lived plasma cells, secrete vaccine protein- specific Abs, increase the serum Ab levels. ➔ Memory B cells are produced ➔ Long-lived plasma cells that can produce Abs for decades, travel to bone marrow niches ➔ CD8+ memory T cells: proliferate rapidly when encountering a pathogen ➔ CD8+ effector T cells eliminate infected cells

Answer 13

o Infectious agents (viruses, bacteria, parasites and their toxins) e.g. polio, tetanus, measles, Hepatitis B etc o Allergy o Cancer

Answer 14

o Injection of allergen in small doses o Gradually increased levels of allergen o Works well e.g. for allergy against grass pollen. o Results in production of blocking antibodies, commonly of IgG4 type. These bind to the allergen and block the otherwise subsequent allergic reaction. o Shifts the T cell response away from TH2 and towards TH1 ➢ Grazax® indicated for disease modifying treatment of grass pollen (Phleum pratense or allergens cross reacting with P. pratense) induced allergic rhinitis with or without conjunctivitis in adults, adolescents and children above the age of 5 years.

Answer 15

Prophylactic cancer vaccines: Gardasil®, Gardasil-9®, Cervarix®, HEPLISAV-B® (HBV-related liver cancer) ➢ Can prevent viral infection which can cause cancer Therapeutic cancer vaccines (TCV): Bacillus Calmette-Guérin (early-stage bladder cancer), Provenge® (prostate cancer) o Help the immune system recognise, target and eliminate cancer cells o Do not target the underlying cause of cancer o Target commonly expressed antigens o Personalised TCVs: ie. autologous therapy: using antigens expressed by a patient’s individual tumor o capable of providing co-stimulatory signals so that the effector cells can attack the tumor cells that lacks such signals.

Answer 16

1. Vaccination with tumor antigen 2. Transport of antigen to lymph nodes 3. Activation of B cells and T-cells. 4.Activated immune cells mount an immune response against the tumor cells. 5. Tumor cell death

Answer 17

Effective vaccines must induce long-lasting protection while being safe and inexpensive A benefit of an effective vaccination program is the ‘herd immunity’ that it confers on the general population.

Answer 18

Herd immunity gives population scale immunity when critical level of vaccination has been achieved * Protection conferred to unvaccinated individuals in a population produced by vaccination of others and reduction in the natural reservoir for infection. * Virus spread stops when the probability of infection drops below a critical level. * This critical level is virus- and population- specific o population % that needs to be vaccinated to achieve herd immunity is 80-85% for polio and 90-95% for measles (highly contagious) * None of the vaccines is or will be 100% effective

Answer 19

-HIV/AIDS -Measels * Effective vaccine for measles is available, but it is heat sensitive, which limits its use in tropical countries *Measles vaccination averted 56 million deaths being between 2000 and 2021. *Even though a safe and cost-effective vaccine is available, in 2021, there were an estimated 128 000 measles deaths globally, mostly among unvaccinated or under vaccinated children under the age of 5 year

Hybridoma & Vaccines Flashcards

(44 cards)