Immunology experiment

Immunology experiment

Scores: 100 pointsReports: 20% (principles, procedures, results, analysis of results)

Attendances: 5%

Examination: 35%

Experiment design: 40%

Preparation: Isolation clothes, reagents

Sanitation

Outlines• Humoral immunity function assay

– Antibody preparation,detection

– Detection Ag orAb in vitro

• Cellular immunity function assay– Separation, proliferation of PBMC

• Non-specific immunity function assay– Phagocytosis of macrophage

• Immune pathology– Hypersensitivity

• Antibody preparation and detection ---- Ⅰ、Ⅱ、Ⅲ、Ⅳ p146

• Agglutination reaction ---- Ⅰ、Ⅱ p2

• Precipitation reaction ---- p6Ⅱ• Antibody formation ablitiy ---- p139Ⅲ• Complement fixation reaction ---- p7Ⅴ• Immunological labeling technology ---- p10Ⅴ

Exp Antibody preparation and ⅠAgglutination reaction (1)

Test 1 Primary immunization

Test 1 Primary immunization

1. Principles:

Test 1 Primary immunization

1. Principles:

Xenoantigen:

Laws of Ab production

SRBCSRBC mousemouse

Test 1 Primary immunization

1. Principles:

Ag

Animals

Serum(Contains Ab)

Subcutaneously, 3 times

Test 1 Primary immunization

2. Methods:Uncoagulated SRBCUncoagulated SRBC

Add 3ml N.S and mixAdd 3ml N.S and mix

2000rpm, 5min2000rpm, 5min

2000rpm, 5min2000rpm, 5min

Discard the supernantDiscard the supernant

Discard the supernantDiscard the supernant

Make 20%Make 20% 、、 25%25% 、、 30%30% 、、40% SRBC solution40% SRBC solution

Take 0.1ml to Take 0.1ml to immunize the mice immunize the mice subcutaneouslysubcutaneously

Add 3ml N.S and mixAdd 3ml N.S and mix

Test 2 Agglutination reaction

Review

Ag

Ab

Ag:Ab reaction

Hv

Lv

Hc

Lc

Ⅰ Nature of Ag:Ab Reactions

Lock and Key Concept: high-specificity

Non-covalent Bonds Hydrogen bonds、 Electrostatic bonds、 Van der Waal

forces、 Hydrophobic bonds

Reversible

Two phases: Binding phase

Visible phase

1. Affinity and Avidity

2. Ag:Ab ratio

Ⅱ Factors Affecting Measurement of Ag/Ab Reactions

Ag: Ab ratio

1. Affinity and Avidity

2. Ag:Ab ratio

3. Physical forms of the antigen

4. Temperature: 37 ~56 ℃ ℃

Ⅱ Factors Affecting Measurement of Ag/Ab Reactions

Agglutination reactions: paticulate Ag

Precipitation reactions: soluble Ag

Effect of temperature

1. Affinity and Avidity

2. Ag:Ab ratio

3. Physical forms of the antigen

4. Temperature: 37 ℃5. PH: 6~8

6. Electrolytes: 0.9% NaCl

Ⅱ Factors Affecting Measurement of Ag/Ab Reactions

Agglutination reaction

Precipitation reaction

Complement fixation reaction

Neutralization reaction

Ⅲ Classical Ag-Ab reactions

• Definition:

Ⅳ Introduction of agglutination reaction

• Classification:Direct agglutination reaction: slide agglutination

tube agglutination

Indirect agglutination reaction

Visible agglomerateParticulate Ag Corresponding Ab

+

Visible agglomerateParticulate Ag Corresponding Ab

+

Direct agglutination reaction

Soluble Ag

Sensitized particulate

Corresponding Ab

+

Visible agglomerateIndirect agglutination reaction

1. Principle: ABO Blood type:

Slide agglutination reaction:

2. Material: Anti-A serum and Anti-B serum

Slide

Needle

Woodstick

Test 1 Assay of blood type (ABO)

+Anti-A serum

OR

Anti-B serum？？

3. Method:

Test 1 Assay of blood type (ABO)

a. Slide

A B

Anti-A Anti-B

b. Pick bloodDisinfect the fingerDisinfect the finger

Stab the fingerStab the finger

Drop one drop of blood into the serumDrop one drop of blood into the serum

c. ReactionMix blood with the serum by woodstickMix blood with the serum by woodstick

d. observation

4. Anticipated results:

Test 1 Assay of blood type (ABO)

Blood typeBlood type

AA

BB

ABAB

OO

5. Attentions:

• Keep the serum seperated

• Disinfection

• Do not confuse the two sides of wooden stick

• Observe the result in time

• Put the blood-stained material in a certain place

Test 1 Assay of blood type (ABO)