Lecture 9/10: Methods/Vaccines

Question 1

ELISA

Answer 1

Enzyme Linked ImmunoabSorbent Assay; Ag/Ab quantification

Question 2

Flow cytometry

Answer 2

IDs specific cell types w/ lasers and fluorescent Abs

Question 3

Agglutination

Answer 3

Cell/molecule clumping when Ab is bound

Question 4

Analyte

Answer 4

Molecule you want to measure

Question 5

Polyclonal Abs

Answer 5

• Mix of Abs from different B cell clones
• Most IgG -> poly clonal response
• Recognize different epitopes on same pathogen
• Change over time for different Ags or increased affinity

Question 6

Monoclonal Abs

Answer 6

• Abs derived from 1 clone
• No affinity maturation or change in antigen
• Consistent response for diagnosis and therapy

Question 7

Creating mAbs

Answer 7

Mice: mix spleen cells w/ cancer cells -> fused immortal hybridoma B cell clone
Humans: isolate desired clones, fuse w/ immortal myeloma

Question 8

Agglutination test

Answer 8

E.g. Coombs test; positive or negative
- Detects Abs to RBC Rh factor, anti-Hu IgG binds mom’s anti-Rh IgG -> RBC agglutination

Question 9

Types of ELISA

Answer 9

Direct: detect/quantify Abs
Indirect (sandwich): detect/quantify Ags

Question 10

Direct ELISA

Answer 10

Ag bound on plate, detected w/ Ab

Question 11

Indirect (sandwich) ELISA

Answer 11

Ag binds surface-coated primary anti-Ag Ab -> wash -> secondary anti-Ag Ab w/ tag gives signal

Question 12

Visualization with tags vs probes

Answer 12

Tags: enyme tag adds colored substrate e.g. HRP; ELISA, IHC
Probes: fluorescent probes allow direct visualization of fluorescence e.g. FITC, Rhodamine; ELISA, immunofluor., flow

Question 13

Serology

Answer 13

Measuring analytes in serum (soluble portion of CLOTTED blood post-centrifuge; plasma = anti-coag. blood post-centrifuge)

Question 14

Titer

Answer 14

Last dilution above background

Question 15

Concentration

Answer 15

Actual Ag amount

Question 16

Absorbance

Answer 16

Dye concentration

Question 17

Lateral flow assay

Answer 17

e.g. at-home COVID test
- Conjugate pad w/ primary labeled Ab
- Test line w/ unlabeled other primary Ab
- Control line w/ secondary Ab
- Flow through generates lines

Question 18

Western Blot

Answer 18

Separate proteins by weight, transfer to membrane, Abs to visualize

Question 19

IHC/IF

Answer 19

• Visualize cell/tissue morph. w/ light or fluorescence microscopy
  1. Prep slide
  2. Add primary mouse anti-Ag Ab
  3. Add labelled secondary anti-Ab HRP
  4. Add substrate for enzyme
  5. Color visual

Question 20

Types of immunization

Answer 20

1. Passive
2. Active

Question 21

Passive immunization

Answer 21

Providing preformed Abs to patient (faster, rabies, antivenom treatment)

Question 22

Active immunization

Answer 22

Using own immune system to make Abs T cells (vaccines)

Question 23

Types of vaccines

Answer 23

1. Live attenuated
2. Conjugate
3. Toxoid
4. Pathogen subunits
5. Dead whole organisms
6. mRNA
7. DNA

Question 24

Live attenuated vaccine

Answer 24

MMR, Rotavirus, Varicella, Influ. nasal, oral polio, BCG
- Pros: long lasting immunity, similar or same immune response, diverse Abs generated
- Cons: can’t give to immunocomp.; pathogenic reversion

Question 25

Conjugate vaccine

Answer 25

Hemophilus influ. type b, pneumococcal conjugate Protein conjugated to polysaccharide -> T dependent response

Question 26

Toxoid vaccine

Answer 26

Diptheria, tetanus Vaccine w/ toxoids similar to pathogen toxins; immune response cross-reacts and neutralizes actual toxin

Question 27

Subunit vaccine

Answer 27

Vax w/ part of pathogen i.e. purified protein, recombinant protein, polysacch., peptides, etc. - Pros: safe, easier quality control - Cons: might not work or be as long lasting

Question 28

Multivalent vaccines

Answer 28

Subunit w/ combo of different subunits in 1 for different strains. Subunit req. proper config. for protection (e.g. RSV failure)

Question 29

Killed whole organism vaccines

Answer 29

Pathogen inactivated w/ chemicals, heat, etc. e.g. hepatitis, rabies, some flu Can be very immunogenic, v. effective

Question 30

Combo vaccines

Answer 30

Multiple methods e.g. DTaP Toxoid: diptheria, tetanus Subunit: acellular proteins

Question 31

mRNA vaccines

Answer 31

Moderna, Pfizer Encapsulated mRNA facilitating DC uptake -> protein expression -> presentation

Question 32

Adenoviral (DNA) vaccine

Answer 32

J&J Disabled adenovirus delivers spike protein DNA; more stable vs mRNA but less immunogenic

Question 33

Adjuvants

Answer 33

Vaccine agents that enhance immune response via innate immunity; stim. DC cytokine secretion - IL-12 -> Th1 diff. -> B support - IL-6 -> B cell prolif. - IFN-gamma -> stim. class switch, Ab response - Upreg. CD28 express.

Question 34

Clinical trial phases

Answer 34

1. Pre-clinical 2. Phase I 3. Phase II/III

Question 35

Pre-clinical phase

Answer 35

Experimental animals for safety/efficacy

Question 36

Phase I trials

Answer 36

Safety + dose escalation; <10 pts often w/ dose escalation, open label, no vulnerable popns

Question 37

Phase II/III

Answer 37

Test for efficacy; more subjects, randomized, controlled, double blind

Question 38

Challenge study

Answer 38

Challenge immunized pts w/ live pathogen; helpful w/ low disease incidence