Module 5 - Immunological Biotechnology

Question 1

What does the immune system do?

Answer 1

• immune system defends us against invasion and infection from pathogens
• cells of immune system circulate in blood and lymph
• lymph is fluid similar to blood but lacking red blood cells (erythrocytes)

Question 2

What are the two types of immunity?

Answer 2

1. natural (innate or non-specific) immunity
   - non-inducible system
   - response doesn’t depend on previous exposure to pathogens or its products
   - includes physical barriers of the body (skin, mucous membranes, tears, perspiration)
   - babies receive passive natural immunity from their mother (transplacental) that develops immediately and lasts for the first few months of the baby’s life
   - includes phagocytes, natural killer (NK) cells (kills body cells that are infected by a microbe), inflammation and fever response
   - not always sufficient to fight off pathogens
2. acquired (adaptive or antigen-specific) immunity
   - APCs (antigen presenting cells) present antigens to antigen-MHC (major histocompatibility complex) complexes on their cell surface
   - depends on the interaction of T-cell receptor (TCR) and foreign antigen presented by APC
   - each TCR has one specificity
   - T-cytotoxic (Tc cells) can directly attack and destroy pathogens
   - T-helper 1 cells (TH 1 cells) act indirectly by secreting cytokines which activate other cells (macrophages) to destroy antigen-bearing cells. This is called cell-mediated immunity
   - T-helper 2 cells (TH 2 cells) interact with antigen-specific B cells and activate them
   - activated B cells divide by mitosis to produce a clone of competent B cells
   - competent B cells differentiate into plasma cells (make antibodies), and memory B cells
   - memory B cells are a part of immunological memory

Question 3

What are polyclonal antibodies?

Answer 3

• made from multiple B cell clones
• recognize different epitopes (antigen surfaces)

Question 4

How to make polyclonal antibodies

Answer 4

• 1). protein of interest is expressed in a suitable system
• protein is usually expressed in denatured form so only some epitopes are present
• protein is mixed with adjuvant
• pre-immune sera is collected for use as a negative control later
  2). protein/adjuvant mix is injected into suitable mammalian host
• mouse, rat, rabbit, guinea pig, hamster, etc.
• whole sera collected (starting at 6 weeks)
• tested by Westerns or other methods for antibody production
• pre-immune sera is the control
• goal is to get high-titer (concentration), high-affinity (strong binding), and high-specificity sera

Question 5

What are monoclonal antibodies?

Answer 5

• monoclonal antibodies are difficult/expensive to make but are useful
• recognizes specific epitopes on a specific protein
• made from a single clone of B cells
• B cells don’t divide on their own in culture, so they are fused with an immortal cancer cell: hybridoma
• steps in protein injection in a suitable host (usually a mouse) is essentially the same as in polyclonal antibodies
• however, instead of blood sera being collected, spleen and lymph nodes are collected
• fusion with tumour cells to make hybridomas
• hybridomas are diluted and plated in cell culture so that there is only one per plate
• screened for desired antibody (Ab) production (ELISA)
• useful clones are selected and maintained

Question 6

What is immunofluorescence?

Answer 6

• fluorescent tag attached to antibody which allows target protein to be identified in cell or tissue staining experiments

Question 7

What are vaccines?

Answer 7

• injection of antigens into individuals
• stimulates long-term immunity which will protect the individual against subsequent infection by pathogen carrying that antigen

Question 8

Types of vaccines

Answer 8

1). live attenuated vaccines
2). inactivated vaccines
3). subunit vaccines
4). toxoid vaccines
5). conjugate vaccines
6). nucleic acid vaccines
7). recombinant vector vaccines

Question 9

What are live attenuated vaccines?

Answer 9

• microbe is weakened by growth in the lab so it doesn’t cause disease

Question 10

What are inactivated vaccines?

Answer 10

• kill microbe with heat, chemicals, or radiation

Question 11

What are subunit vaccines?

Answer 11

• 1-20 epitopes or antigens vs. an entire microbe
• can grow the microbe and then break it apart to create antigens
• can express antigens separately using recombinant DNA techniques

Question 12

What are toxoid vaccines?

Answer 12

• toxin secreted by microbe (vs. microbe itself) used for vaccination
• treat toxin with formalin (formaldehyde + sterile water) to inactivate toxins

Question 13

What are conjugate vaccines?

Answer 13

• specifically for infants and young children
• polysaccharide coating on certain pathogenic bacteria can hide the antigen from the immune system

Question 14

What are nucleic acid vaccines?

Answer 14

• introduce genes as ‘naked DNA’ in patient
• genes will express antigenic proteins in patient
• patient manufactures antigens that stimulate his/her own immune response

Question 15

What are recombinant vector vaccines?

Answer 15

• delivery vector as carrier instead
• delivery vector might be attenuated virus or bacteria with harmful genes as ‘DNA of interest’