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Antigen-Antibody ReactionSerial Dilution Technique

Laboratory 2

Assist. Prof. SuwinWongwajana, M.D., M.Sc.

Dept.of Microbiology Fac.of Medicine, KKU

For 2nd Year Pharmaceutical Science Student

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1 2 3 4 5 6

D.W. 05.

ml

0. 5

ml

12 14 1Dilution : : : 8 116 132: :

control

Color water

0. 5

ml

0. 5

ml

0. 5

ml0.

5ml

0. 5

ml

Serial dilutionSerial dilution

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N1 = Dilution (concentration) of the 1st tube V1 =Volume that pipette from the 1st tubeN2 = Dilution (concentration) of the 2nd tube

V2 = Total volume of the 2nd tube(V2 = V1 + initial volume of the 2nd tube)

Calculation of Calculation of DilutionDilutionNN11VV11 = N = N22VV22

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14: ?

2 3

05.ml

N1 N

2

V1

V2 = 0.5+0.5 =1

N2 = 1/4 x 1/2 =1/81

D.W. 0 5.

ml

2N = N 1 1x V2

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Demonstration - On the bench near the windowsAnswer:Answer: Ab titerAb titer = ?= ?

ExperimentExperiment 1. 1. Tube Tube precipitation testprecipitation test

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1 2 3 4 5 6

Diluent0.

5ml

0Serum 5. ml

0. 5

ml

1 1 1 1Antigen 1 1 drop

1 :2 1 :4 1 : 8 116 132: :

control

0. 5

ml

0. 5

ml

0. 5

ml0.

5ml

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Ab Titer = 18 8: or

precipitate

12 14Dilution : : 18 116 132: : :

control

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ExperimentExperiment 2 2.. ImmunodiffusionImmunodiffusion1

2

3

4

5

6

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Ag1Ag2

Ag3Ag1

Ag2

Ag3

Ab

-Practice applying the Ag and Ab into the wells -Observe the precipitin band (from your Gr. tutor)

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- Perform the finger stick on your friend’s finger. - Use sterile technique – to prevent the infection- Wipe the finger with 70% Alcohol , stick the finger with lancet , drop the blood onto the glass slide.- Discard the lancet, cotton, etc. to the appropriate receptacles.

Experiment Experiment 3 . 3 .Blood groupingBlood grouping

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Blood drop from the finger

Anti-A

Anti-B

N NNNNNNNNNNNN What is the

blood gr. ?A

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Agglutination

No Agglutinatio

n

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Demonstration 1. Single radial

immunodiffusion (SRID)2. Immunoelectrophoresis (IEP)3. Immunodiffusion (ID)4. Tube precipitation

5. Enzyme-linked immunosorbent assay (ELISA)

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- Enzyme linked immunosorb- Enzyme linked immunosorb ent assay ent assay (ELISA) (ELISA)

By...By... Assistance Professor Suwin Wongwajana,MD. Assistance Professor Suwin Wongwajana,MD.

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NNNNNN N NNNNN( ) NNNNNN N NNNNN( )

96 8wells plate ( row , 1 2 )

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Indirect ELISA (For detec Indirect ELISA (For detec tion of antibody) tion of antibody)

1. Coat plate wi 1. Coat plate wiNN N NNNNNNNN N NNNNNN

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2 2WashWash

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YYY Y 3. Add specimens ( 3. Add specimens (

with Ab, or No Ab) with Ab, or No Ab)YYY Y

No specific AbY

YYY

with specifi c Ab

(Y)Y

YYY

YY

YY

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No specific AbY

with specificAb

(Y)

4 4WashWash

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-4. Add conjugate (anti h -4. Add conjugate (anti h = ) = ) Y

EE EEY

EE EE

YEEEEYEEEE

YEE EEY

EE EE

YEEEEY

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with specificAb

No specific Ab(Y)

5. 5.WashWash

YYEEEEYEEEE

EE EE

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Human-IgG

Yimmunized

-Rabbit anti hum an IgG

Y

=Ag

YY

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YYEEEEYEEEEEE EEYYEEEEYEEEE

EE EE

6 . Add substrate 6 . Add substrate( )( )

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YYEEEEYEEEE

EE EE

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Absorbance (O.D.)

MeasurementIntensity of the color

developed, Correlated with the concentration of the

unknown Ab in the specimen

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ELISA READER

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N NNN NNNN

NNNNNNNNNNNN

ImmunofluorescenceImmunofluorescence(IF)(IF)

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Direct Immunofluorescenc Direct Immunofluorescenc e (For detection of antigen e (For detection of antigen

))

CD4

CD3

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4CD

3CD

-Anti CD4 = NNNNNNNNNNN NNN

+ F*

*F*

*

F*F*

F*

*F*

*

*F* *

*F*

*

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4CD +ve

Fluorescent Micr oscope

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CD4

CD8

CD8 CD8

CD4

CD4

CD4CD4

CD4

CD8

CD8 CD8

CD4

CD4

CD4CD4

CD4

CD8

CD8 CD8

CD4

CD4

CD4CD4

CD4

CD8

CD8 CD8

CD4

CD4

CD4CD4

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Indirect Immunofluorescenc Indirect Immunofluorescenc e (For detection of antigen) e (For detection of antigen)

F*

*F

F* F**

F*

Specific Ab

F**

F*

Conjugate

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F**

F*F**

F*

-Rabbit anti CA Ag

F**

F*

- Mouse anti rabbit = *F

CA Ag

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What should you do, What should you do, todaytoday

2. Perform blood grouping. 3 . Practice applying the

well in slide immunodiffusion.4. Observe all the demonstration points.

1 . Practice serial dilution by sterile technique.

5. Conference today start at 3 30 pm . - Quiz and discussion The time given for the lab

hour until 3 30. pm