IA. General Microbiology | 19. Antigen-antibody reactions used in laboratory.

Question 1

I. Basics
1. What is serology?

Answer 1

Antigen + antibody binding in vitro
= forming detectable complex

Question 2

I. Basics
2. How do we use serology?

Answer 2

• Can investigate unknown antigens with known antibodies , or presence of antibodies with known antigens, or qualitative and quantitative detection of antibodies and antigens
• Used if detection of the antigen is urgent (life-threatening, epidemiological importance) or if cannot be cultivated on artificial culture media and/or requires long or expensive cultivation
  methods

Question 3

II. Antigen-antibody reactions used in lab
1. What are the 2 main types of Antigen-antibody reactions used in lab?

Answer 3

1. Ag-Ab binding with visible result
2. Indirect detection of Ab-Ag binding

Question 4

II. Antigen-antibody reactions used in lab
1. What are the 2 main types of Antigen-antibody reactions used in lab?

Answer 4

1. Ag-Ab binding with visible result
2. Indirect detection of Ab-Ag binding

Question 5

II. Antigen-antibody reactions used in lab
2. What are the 2 reactions types of Ag-Ab binding that give visible result?

Answer 5

1. Agglutination
2. Precipitation

Question 6

II. Antigen-antibody reactions used in lab
3A. What is Agglutination in Ag-Ab binding?

Answer 6

cellular antigen and specific antibody bonds causing of
the cells

Question 7

II. Antigen-antibody reactions used in lab
3B. What are the 3 types of agglutination caused by Antigen-antibody binding?

Answer 7

1. Qualitative: Slide- agglutination
2. Quantitative: tube agglutination (Widal’s type reaction)
3. Coagglutination

Question 8

II. Antigen-antibody reactions used in lab
3C. What are the features of Slide- agglutination?

Answer 8

• Reagent (Ab)+ saline + bacterium(Ag)= clumps (positive) or homogenous (negative)
• e.g. serotyping E.Coli

Question 9

II. Antigen-antibody reactions used in lab
3D1. What are the features of tube- agglutination?

Answer 9

Quantitative: tube agglutination (Widal’s type reaction) (i.e. Dilution row of erum(Ab) + known Ag → quantity of antibodies present in the patient’s serum against antigen
- Titer: highest dilution (lowest concentration) where agglutination occurs
- Seroconversion: at least 4x increase in the antibody titer between serums obtained with 2-4 weeks different
- Gruber-Widal reaction= Salmonella typhi, Weil-Felix= Rickettsia, Wright= Brucella

Question 10

II. Antigen-antibody reactions used in lab
3D2. What is Titer in tube- agglutination?

Answer 10

Titer: highest dilution (lowest concentration) where agglutination occurs

Question 11

II. Antigen-antibody reactions used in lab
3D3. What is Seroconversion in tube- agglutination?

Answer 11

at least 4x increase in the antibody titer between serums obtained with 2-4 weeks different

Question 12

II. Antigen-antibody reactions used in lab
3E1. What are the features of Coagglutination?

Answer 12

Smaller antigen/ or antibody was absorbed onto a surface of a larger particle (Such as latex bed)

S. Aureus’s Protein A binds to the Fc region of immunoglobulins → specific Fab region remains
free, can bind Ag-s
○ Latex agglutination: Immunoglobulins are bound to latex particles (e.g. Streptococcus serotyping)

Question 13

II. Antigen-antibody reactions used in lab
3E2. What is Latex agglutination (Coagglutination)?

Answer 13

Immunoglobulins are bound to latex particles (e.g. Streptococcus serotyping)

Question 14

II. Antigen-antibody reactions used in lab
4A. Describe Precipitation in Antigen-antibody reactions?

Answer 14

dissolved / and specific antibody binds → visible complex formation= precipitate

Question 15

II. Antigen-antibody reactions used in lab
4B. What are the 3 major types of precipitation caused by Antigen-antibody binding?

Answer 15

1. Precipitate in liquid medium
2. Precipitate in agar gel– immune diffusion
3. Gel precipitation: double immunodiffusion

Question 16

II. Antigen-antibody reactions used in lab
4C. Describe precipitation in liquid medium

Answer 16

• Qualitative disk precipitation: Ag and Ab diffuse towards each other, precipitation occurs where their amount is equal → zone of equivalence: ag-ab binding is optimum and precipitation shows
• Quantitative: determination of toxin concentration in a solution with known antitoxin (Ab against toxin containing serum) (e.g. flocculation tests for syphilis diagnostics)

Question 17

II. Antigen-antibody reactions used in lab
4C. Describe precipitation in agar gel– immune diffusion

Answer 17

Elek’s-test (detection of toxin production)
• Corynebacterium diphteriae from patient’s sample is
placed on an agar plate
• filter paper infused with anti-Corynebacterium
diphteriae toxin antibodies (shortly antitoxin) is placed perpendicularly
• if the sample strain produces the toxin (the antigen), it diffuses into the agar, binds to the antibodies and a precipitation line appeares

Question 18

II. Antigen-antibody reactions used in lab
4D. Describe Gel precipitation: double immunodiffusion

Answer 18

• both substances diffuse
• precipitation line where they meet

Walls are cut in agar gel, poured in glass slide or petri dish→ antiserum w/ antibodies are poured in central wall→ different antigens added → lines of precipitation are formed at sites of combination of ab and antigen
(Double immunodiffusion is an agar gel immunodiffusion.

It is a special precipitation reaction on gels where antibodies react with specific antigens forming large antigen-antibody complexes which can be observed as a line of the precipitate.

In double immunodiffusion, both the antibody and antigen are allowed to diffuse into the gel.

After application of the reactants in their respective compartments, the antigen and the antibody diffuse toward each other in the common gel and a precipitate is formed at the place of equivalence. )

Question 19

II. Antigen-antibody reactions used in lab
4E. Can you name other precipitation methods?

Answer 19

single radial immunodiffusion, countercurrent electrophoresis, immunoelectrophoresis

Question 20

II. Antigen-antibody reactions used in lab
5. What are the 3 types of Indirect detection of Ab-Ag binding?

Answer 20

1. Complement fixation
2. Virus or toxin neutralization
3. Labeling based on antigen/antibody

Question 21

II. Antigen-antibody reactions used in lab
6A. What are the features of complement fixation?

Answer 21

• Components: antigen, antibody (patient’s own serum), complement (titrated serum of guinea pig), sheep RBC suspension, hemolysin: anti-sheepRBC antibody from immunized rabbit
• E.g. Wasserman reaction→ screening for syphilis
• Positive= no hemolysis, RBC-s settle down
• Negative= hemolysis, no visible clot

Question 22

II. Antigen-antibody reactions used in lab
6B. What are the components of Complement fixation?

Answer 22

Components: antigen, antibody (patient’s own serum), complement (titrated serum of guinea pig), sheep RBC suspension, hemolysin: anti-sheepRBC antibody from immunized rabbit

Question 23

II. Antigen-antibody reactions used in lab
6C. Explain the positive and negative results of complement fixation?

Answer 23

• Positive= no hemolysis, RBC-s settle down
• Negative= hemolysis, no visible clot

Question 24

II. Antigen-antibody reactions used in lab
6D. Give an example of complement fixation?

Answer 24

E.g. Wasserman reaction→ screening for syphilis

Question 25

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding
7. What are the features of Virus or toxin neutralization?

Answer 25

• Neutralizing antibody: Ab capable of inhibiting the activity of its specific antigen (toxin/virus) - Detection of antibody or antigen in patient’s serum

Question 26

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding
8A. What are the 4 types of Labeling based on antigen/antibody?

Answer 26

1. Immunofluorescence assay (IFA)
2. Radioimmunoassay (RIA)
3. ELISA (Enzyme linked immunosorbent assay)
4. Western Blot

Question 27

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding - Labeling based on antigen/antibody
8B1. What are the features of 1. Immunofluorescence assay (IFA)?

Answer 27

a. Ig labelled with fluorescent dye (fluorescein-isothiocyanate) binds to specific antigen and complex emits UV light
b. Detection of antigens on the surface of bacteria or in slides
c. Direct(DIF) in detection of chlamydia trachomatis
d. Indirect (IDIF): first unlabeled Ab interacts with antigen; second, labelled ab reacts with first ab (more sensitive)

Question 28

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding - Labeling based on antigen/antibody
8C. What are the features of Radioimmunoassay (RIA)?

Answer 28

• Antibody detection: solid phase immunoassay
• Antigen labelled with radioisotope + patient’s serum
• Amount of ab proportional to amount of fixed radioisotope
• Unlabelled Ab in patient’s serum compete with the same type, labelled antibody for antigens
• Very specific and sensitive, but complicated, expensive, and
  dangerous

Question 29

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding - Labeling based on antigen/antibody
8D. What are the features of ELISA (Enzyme linked immunosorbent assay)?

Answer 29

• antigen-specific antibodies placed in the wells of the ELISA plate (adsorbed to its surface)
• sample is added to each well – if antigen is present in the sample, the antibody captures it (binds to it)
• Another, enzyme-linked, antigen specific antibody is added to the sample
• Transparent substrate is added to the well, which then can be converted into a colored pruduct by the enzyme
• As we rinse the well between each steps, the color change is only seen if the antibodies were able to bind to the antigen (if the antigen was present)

• E.g. Borrelia Burgdorferi (Lyme-disease), HCV, HIV

Question 30

II. Antigen-antibody reactions used in lab - Indirect detection of Ab-Ag binding - Labeling based on antigen/antibody
8E. What are the features of Western Blot?

Answer 30

• Antigens (viral proteins) separated by electrophoresis, based on weight and charge
• Transfer (blotting) onto nitrocellulose membrane
• Patient’s serum is added→ antibodies bind to the specific antigens
• Detection: enzymes labelled anti-human Ig and substrate