serological diagnosis of viral infections fixation test (cft) iii – based on application of...
TRANSCRIPT
![Page 1: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/1.jpg)
SEROLOGICAL DIAGNOSIS OF VIRAL
INFECTIONS:
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
TYPES OF SEROLOGICAL REACTIONS
![Page 2: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/2.jpg)
SEROLOGICAL REACTIONSSEROLOGICAL REACTIONS
Ag-Ab reactions used for the detection of unknown Ag or Ab, in vitro
In virology:
Viral (direct) diagnosis:- detection of viral antigens
Serological (indirect) diagnosis:- detection of specific antiviral antibodies
![Page 3: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/3.jpg)
Ø Qualitative detection of total specific antiviral antibodies
Ø Quantification of total specific antiviral antibodies (titer of Abs)
1/2 1/4 1/8 1/16 1/32 1/64
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
![Page 4: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/4.jpg)
Ø Detection of Ab classes (qualitative and quantitative)
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
![Page 5: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/5.jpg)
Ø Detection of Ab synthetized to each viral protein (Ag) of specific molecular weight –serological profile
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
![Page 6: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/6.jpg)
Ø Determination of specific antibody avidity
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
POSSIBILITIES OF SEROLOGICAL DIAGNOSIS
![Page 7: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/7.jpg)
C l a s s i c a lM
o d e r nTypes of serological reactionsTypes of serological reactions
I – detection of Ab that block biological functions of the virusNeutralization testHaemagglutination Inhibition Test
II – based on biological activity of Abs in complex with AgsImmunoagglutinationComplement Fixation Test (CFT)
III – based on application of labeled antibodiesEIA (ELISA)ImmunofluorescenceRIA
IV – determination of serological profileWestern blot
![Page 8: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/8.jpg)
I – Ab that block biological functions of the virus
Neutralization Test neutralizing Ab(Abs that block viral infectivity)
viral suspension
neutralizing Abs bind to viral anti-receptors
patient’s serum
inoculation in the cell culture
No CPE!!
![Page 9: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/9.jpg)
Titer of Ab reciprocal value of the highest dilution of patient’s serum that gives positive reaction
Multiple (twofold, fourfold, tenfold…) serial dilutions of patient’s serum:
Titer of neutralizing Ab is ?
![Page 10: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/10.jpg)
INHIBITION OF HAEMAGGLUTINATION
RBCPatient’s serum
Haemagglutination Inhibition Test neutralizing Ab(Abs that block viral
haemagglutination)
Virus
I – Ab that block biological functions of the virus
![Page 11: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/11.jpg)
Haemagglutination Inhibition Test
Titer of Ab reciprocal value of the highest dilution of patient’s serum that gives positive reaction
Multiple (twofold, fourfold, tenfold…) serial dilutions of patient’s serum:
Titer of haemagglutinating Ab is?
![Page 12: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/12.jpg)
Haemagglutination Inhibition TestProcedure:
Incubation at 37°C
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl virus
+
50 µl RBC
50 µl RBC
50 µl virus
+
50 µl RBC
1:256
50 µl saline
+50 µl
1:128
50 µl saline
+50 µl
1:64
50 µl saline
+50 µl
1:32
50 µl saline
+50 µl
1:16
50 µl saline
+50 µl
1:8
50 µl saline
+50 µl
1:4
50 µl saline+
50 µl serum
1:2
50 µl saline+
50 µl serum
Positive Control
50µl saline
Negative Control
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10
1
2
3
4
![Page 13: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/13.jpg)
Immunoagglutination
Indirect immunoagglutination - qualitative
II – based on biological activity of Abs in complex with Ags
- Reaction between corpuscular Ag and specific Ab, manifested as forming of agglutinates
![Page 14: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/14.jpg)
Immunoagglutination
Indirect immunoagglutination - qualitative
![Page 15: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/15.jpg)
Complement Fixation Test (CFT)
II – based on biological activity of Abs in complex with Ags
= antigen = antibody = complement
First step Second step Result
No haemolysis
Haemolysis
Sensitized RBC
![Page 16: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/16.jpg)
Complement Fixation Test (CFT)
1/8 1/16 1/32 1/64
Titer of Ab reciprocal value of the highest dilution of patient’s serum that gives positive reaction
Multiple (twofold, fourfold, tenfold…) serial dilutions of patient’s serum:
Titer of Ab is?
![Page 17: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/17.jpg)
ELISA – Enzyme Linked ImmunoSorbent Assay
III – based on application of labeled antibodies
![Page 18: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/18.jpg)
ELISAELISA
![Page 19: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/19.jpg)
ELISAELISA
![Page 20: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/20.jpg)
Western blot as serological reaction: confirmation test(HIV)
IV – determination of serological profile
Serological profile – presence of Ab specific for different viral Ag
![Page 21: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/21.jpg)
Western Blot for HIV
HIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive
üü At least 1 Ab for products of all 3 major genes (At least 1 Ab for products of all 3 major genes (33))
üü (CDC) Two Ab for any of the 3 major Ag: (CDC) Two Ab for any of the 3 major Ag: p24, gp41, gp120/160p24, gp41, gp120/160 (2)
3 genes:3 genes:
--GAGGAG
--POLPOL--ENVENV
POSITIVE
![Page 22: SEROLOGICAL DIAGNOSIS OF VIRAL INFECTIONS Fixation Test (CFT) III – based on application of labeled antibodies EIA (ELISA) Immunofluorescence RIA IV – determination of serological](https://reader031.vdocuments.site/reader031/viewer/2022022602/5b5468927f8b9aa40e8d01eb/html5/thumbnails/22.jpg)
AVIDITY OF ANTIBODIESAVIDITY OF ANTIBODIES
Ag
At
Avidity – strength of bond between Ab and Ag
* low-avidity antibodies (primary immune response)
* high-avidity antibodies (memory immune response)