exp Ⅱ agglutination reaction and antibody preparation (2)

ExpⅡ Agglutination reaction and Antibody preparation (2)

Test 1 Agglutination reaction

1.Principle:

Tube agglutination reaction

Ag-Ab reaction

Agglutination reaction

Slide agglutination reaction

Qualitative Semi-Quantitative


1.Principle:


1) Slide agglutination reaction:

Known Ag + Unknown AbAgglutination

turbid?2) Tube agglutination reaction:

Quantitative Ag + Serially diluted Ab

Different degree of agglutination

YES

NO

Determine the titer of Ab

1.Principle:

What is the titer of Ab?

The titer is customarily reported as the reciprocal of the highest dilution of Ab that causes an obvious agglutination.


2. Experiments

Ⅰ Tube agglutination test

Ⅱ Slide agglutination test



1. Material: Inactivated typhiod bacteria (7×108/ml), anti-serum

2. Method:

0.5 0.50.50.50.50.5 0.5 0.5 0.5

1:20 1:40 1:80 1:160 1:320

0.5Discard

1 2 3 4 5

0.1

6

0.9N.S

Anti-H Serum

Control

0.5ml 0.5ml 0.5ml 0.5ml 0.5ml①①

②②

③③

④④

Inactivated typhiod bacteria

0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml

Dilution ratio of Ab

Shake56℃, 1~2h

3. Attentions

Sign the tube Use the dropper tube correctly Mix thoroughly T: 56℃ Record the time Take the tube carefully


4. Anticipated results

+Very small agglutinationturbid

-No agglutinationSame as control

++Small and depositing partlyObvious turbid

+++Large and depositing mostlySlightly turbid

++++Large and depositingClear

Degree of agglutinationSedimentSupernantant


Sediment

Agglutination degree

The highest dilution ratio

Titer of the anti-serum


1. Material: Staphylococci

Inoculation loop

2. Method:

1 2

Anti serum1

a. Slide

b. Bacteria

c. ReactionMix bacteria with the serumMix bacteria with the serum

d. observation

Anti serum2

3. Anticipated results:

++ --


4. Attentions:

Sterile operation


Test 2 Secondary 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 immTake 0.1ml to immunize the mice subcunize the mice subcutaneouslyutaneously

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

exp Ⅱ agglutination reaction and antibody preparation (2)

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