Name: PowerPoint Presentation
Uploaded: 2022-04-24
Duration: 51 min 10 s
Description: Faculty of Veterinary Medicine Department of virology General Program – Virology – 3 rd Level Students.

. Faculty of Veterinary Medicine Department of virology General Program – Virology – 3 rd Level Students. 

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Faculty of Veterinary Medicine Department of virology General Program – Virology – 3 rd   Level Students

Principle:  Antigen antibody reaction  Antibody is labeled by fluorescent dye  It absorb the UV rays(short wave length)  and release it as a long wavelength dye ..seen by naked eye) Dyes as : FITC yellowish green  Rhodamine dye  orange Seen   under   : fluorescent microscope  High pressure mercuric lamb  emit UV rays  Uses  :  1- detect virus in cell culture  2- detect site of virus replication

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Direct IF                                                              Indirect IF

Faculty of Veterinary Medicine Department of virology General Program – Virology – 3 rd Level Students. 

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1- frozen tissue section 2- fixation by  acetone  for 10 mins 3- add labeled ab and incubate at 37 / 30 min in humid place Wash 1% PBS Jar PBS for 10 min Blot dry Drop of glycerol  Cover slip  examine

1 , 2  the same. 3- add  un-labeled  ab  and incubate at 37 / 30 min in humid place Wash  three times by 1% PBS Add labeled  antiglobulin (  antispp   ab )  , wash  examine

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Indirect IFT Is better than Direct IFT???

1- More economic than labeling every anti viral   ab  when studying more than one virus 2- more sensitive    antispp   Ab  conserved fc  porion

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Result :      +ve fluorescence -ve no fluorescence

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Principle: Ag + Ab (labeled with Enzyme ) E (no color)+ substrate   product (color)  Naked eye or ELISA reader spectrophotometer  Enzyme : horse raddish peroxidase Substrate  : OPD blue Microtiter plate flat bottom Stop reaction by  5 N . H 2 SO 4  (stopping solution) yellow color

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Direct  Sandwich Antibody capture ELISA Detect unknown Ag

Indirect Antigen capture ELISA Detect unknown AB

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Substrate
Capture
antibody
Capture Assay
"Sandwich"

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Substrate
Secondary
antibody
conjugate
Primary
antibody
Indirect Assay

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3
Viral antigen in
patient's sarnple
Enzyrne—labeled
antiviral antibodies
Substrate

Ab coated  mictotiter  plate blocking by : 1% BSA, gelatin , casein,  fetal calf serum,  Add tested ag Incubate 37/ 1  hr Wash by PBS – tween 20 three times Add labeled ab ( conjugate) Incubate 37/ 1  hr Add substrate OPD Stop  rxn  by  sulphoric  acid  bcz   rxn  is reversable

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Ag coated mictotiter plate blocking by : 1% BSA, gelatin , casein,  fetal calf serum,  Add tested ab Incubate 37/ 1 hr Wash by PBS – tween 20 three times Add labeled anti-ab ( conjugate) Incubate 37/ 1 hr Add substrate OPD Stop rxn by sulphoric acid bcz rxn is reversable

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Microplate ELISA for HIV antibody: coloured wells indicate reactivity

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Primary cntitxxiv
conugate
Ag
Direct ELISA
Capture
SandwiCh ELISA
Secondary
antibody
Indirect ELISA
Inhibitor
antigen
Competitive ELISA

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The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody.  The reference antigen is pre-coated on a multi-well plate and  sample is pre-incubated with labeled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen.  This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.

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