Exp ? 1. Enzyme-linked Immunosorbent Assay(ELISA) 2. Complement fixation test (CFT) - PowerPoint PPT Presentation

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Exp ? 1. Enzyme-linked Immunosorbent Assay(ELISA) 2. Complement fixation test (CFT)

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Title: Exp ? 1. Enzyme-linked Immunosorbent Assay(ELISA) 2. Complement fixation test (CFT)


1
Exp ? 1. Enzyme-linked Immunosorbent
Assay(ELISA)2. Complement fixation test (CFT)
2
1. Enzyme-linked Immunosorbent Assay (ELISA)
3
Immunological labeling techniques
  • Definition
  • Ag-Ab reactions are combined with labeling
    techniques. Known Ag or Ab is labeled with
    radioisotope, enzyme or fluorescein and unknown
    Ab or Ag are indirectly detected by labeled
    molecules.
  • Classification
  • Radioimmunoassay(RIA) 131I,32P
  • Immunofluorescence technique FITC,PE
  • Enzyme Immunoassay HRP,AP

4
Immunological labeling techniques
3. Labeling techniques
Direct labeling techniques Each Ag or Ab
Indirect labeling techniques Secondary Ab
5
Isotype of antibody
Immunize Rabbit
Mouse IgG1
Rabbit anti-mouse IgG1 Secondary Ab
6
Immunofluorescence technique
7
Enzyme ImmunoAssay (EIA)
  • Ag-Ab reactions with enzyme-labeled Ag or Ab.
  • horseradish peroxidase (HRP),alkaline phosphatase
    (AP)
  • Characteristics
  • High specificity Ag-Ab reaction
  • High sensitivity enzyme catalytic reaction (pg
    level)
  • Qulitative or quantitative assay Color change or
    OD value
  • Classification
  • ELISA Soluble Ag or Ab
  • Immunochemistry Ag in tissues or in the surface
    of cells

8
Immunohistochemistry
9
Enzyme-Linked ImmunoSorbent Assay (ELISA)
1.Definition
Unknown Ab or Ag in blood or culture medium are
detected by enzyme-labeled Ag or Ab.
2.Classification
  • Indirect ELISA
  • Known Ag, enzyme-labeled secondary Ab
  • Unknown Ab
  • Sandwich ELISA
  • Known Ab, enzyme-labeled Ab
  • Unknown, soluble Ag
  • Competitive ELISA
  • Known Ab or Ag, enzyme-labeled Ab or Ag
  • Unknown Ag or Ab

10
Enzyme-Linked ImmunoSorbent Assay (ELISA)
3. Principles (Indirect ELISA for example)
11
(No Transcript)
12
Experiment Assay of hemolysin by Indirect
ELISA Qualitative assay
13
Materials
  • Defribinated SRBC Ag
  • Rabbit anti-SRBC Ab(hemolysin) Primary Ab
  • HRP-labeled goat anti-rabbit IgG Secondary Ab
  • Coating buffer0.05M pH9.6 bicarbonate buffer
  • Washing buffer0.01M PBS(pH7.4) containing 0.05
    Tween 20
  • Substrate buffer pH5.0 phosphate-citrate buffer
    solution
  • Substrate solutionOPD 10mg,Substrate buffer
    25ml,30 H2O2 40µL
  • Microwell plate

14
Methods
Defibrated SRBC
Add 3mL N.S and mix
2000rpm,5min
1. Preparation of Ag
Add 3mL N.S and mix
2000rpm,5min
Take 2mL packed SRBC
Add 2mL DDW and shatter RBC
Dilute with coating buffer in a ratio of 1400
Add 100µL of Ag to each well of ELISA plate
2. Coating Ag
4?,in a humidified box, 18h
15
Methods
Discard the Ag solution in ELISA
plate(4wells/group)
Wash the plate 3 times(3min each time)
3. Add test serum
1
2
3
4
Sign and add 100µL of solution to each well
Negative
Blank
Positive
Test
37? for 45min in a humidified box
Discard the solution in ELISA plate
4. Add secondary Ab
Wash the plate 3 times(3min each time)
Add 100µL of HRP-labeled secondary Ab to each well
37? for 30min in a humidified box
Discard the solution in ELISA plate
5. Showing color
Wash the plate 3 times(3min each time)
Add 100µL of substrate solution to each well
Show color in dark for10min
6. Observe the result
16
Anticipated results
  • Positiveyellow
  • Negativeblank

1 2 3 4
Neg
Neg
Pos
Pos
17
Attentions
  • Wash thoroughly and avoid cross-contamination
  • Add samples in turn and no bubbles in the bottom
    of the wells
  • Coating and incubation should be performed in
    humidified box

18
2. Complement fixation Test(CFT)
19
Definition
  • Complement fixation reaction
  • Ag-Ab reaction in the presence of
    complement and with SRBC and hemolysin (anti-SRBC
    Ab) as an indicator system.

20
Principle
  • Two systems, five components in CFT
  • Test system known Ag or Ab, unknown Ab or Ag and
    quantitative complement.
  • Indicator system SRBC and hemolysin

21
AgUnknown serum
Complement
SRBChemolysin
1
2
1
2
Step 1 Formation of Ag-Ab complex
Step 2 Complement fixation and exhaustion
Step 3 Lysis of SRBC
22
Ab Positive
No Ab Negative
Ag
Ag
Ag
Ag
Y

23
Material
  • Ag Lytic products from typhiod
  • Test serum 56?,30min
  • Complement from guinea pig
  • 2 SRBC
  • Hemolysin

24
Methods
Serum control
Ag control
Complement control
SRBC control
Test
1
2
3
4
5



0.2mL
Test serum
0.2mL
Ag



0.2mL
0.2mL
Complement

0.2mL
0.2mL
0.2mL
0.2mL
N.S

0.2mL
0.4mL
0.8mL
0.2mL
Shake,incubate in water bath at 37? for 15min

Hemolysin
0.2mL
0.2mL
0.2mL
0.2mL
0.2mL
0.2mL
SRBC
0.2mL
0.2mL
0.2mL
Shake,incubate in water bath at 37? for 15min
25
Anticipated results
1.Hemolysis clear, red No hemolysis
turbid, ruddy
Hemolysis
No hemolysis
2.
Test Serum control Ag control Complement control SRBC control
? Hemolysis Hemolysis Hemolysis No hemolysis
26
Attentions
  • Shake SRBC before use
  • Pipettes for different reagents should not be
    confused
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