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Exp Ⅴ 1. Enzyme-linked Immunosorbent Assay ( ELISA) 2. Complement fixation test (CFT) PowerPoint Presentation
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Exp Ⅴ 1. Enzyme-linked Immunosorbent Assay ( ELISA) 2. Complement fixation test (CFT). 1. Enzyme-linked Immunosorbent Assay ( ELISA). Immunological labeling techniques. Definition:

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immunological labeling techniques
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
immunological labeling techniques1
Immunological labeling techniques

3. Labeling techniques:

Direct labeling techniques: Each Ag or Ab

Indirect labeling techniques: Secondary Ab

slide5

Isotype of antibody:

Immunize Rabbit

Mouse IgG1

Rabbit anti-mouse IgG1

Secondary Ab

e nzyme i mmuno a ssay eia
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
e nzyme l inked i mmuno s orbent a ssay elisa
Enzyme-Linked ImmunoSorbent Assay (ELISA)

1.Definition:

Unknown Ab or Ag in blood or culture medium are detected by enzyme-labeled Ag or Ab.

  • 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

2.Classification:

e nzyme l inked i mmuno s orbent a ssay elisa1
Enzyme-Linked ImmunoSorbent Assay (ELISA)

3. Principles (Indirect ELISA for example):

experiment assay of hemolysin by indirect elisa qualitative assay
Experiment:Assay of hemolysin by Indirect ELISA——Qualitative assay
materials
Materials:
  • Defribinated SRBC —— Ag
  • Rabbit anti-SRBC Ab(hemolysin) —— Primary Ab
  • HRP-labeled goat anti-rabbit IgG —— Secondary Ab
  • Coating buffer:0.05M pH9.6 bicarbonate buffer
  • Washing buffer:0.01M PBS(pH7.4) containing 0.05% Tween 20
  • Substrate buffer: pH5.0 phosphate-citrate buffer solution
  • Substrate solution:OPD 10mg,Substrate buffer 25ml,30% H2O2 40μL
  • Microwell plate
methods
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 1:400

Add 100μL of Ag to each well of ELISA plate

2. Coating Ag:

4℃,in a humidified box, 18h

methods1

Discard the Ag solution in ELISA plate(4wells/group)

Methods

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

Blank

Negative

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

anticipated results
Anticipated results
  • Positive:yellow
  • Negative:blank

1 2 3 4

Neg

Neg

Pos

Pos

attentions
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
definition
Complement fixation reaction:

Ag-Ab reaction in the presence of complement and with SRBC and hemolysin (anti-SRBC Ab) as an indicator system.

Definition
principle
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
slide21

1

2

Ag+Unknown serum

Complement

SRBC+hemolysin

1

2

1

2

Step 1

Formation of Ag-Ab complex

Step 2

Complement fixation and exhaustion

Step 3

Lysis of SRBC

slide22

Patient’s

serum

Y

Y

Y

Y

Y

Y

Y

Y

Ab

Positive

No Ab

Negative

Ag

Ag

Ag

Ag

Y

material
Material
  • Ag: Lytic products from typhiod
  • Test serum: 56℃,30min
  • Complement: from guinea pig
  • 2% SRBC
  • Hemolysin
methods2

Serum control

Ag control

Complement control

SRBC

control

Test

Methods

1

2

3

4

5

… …

… …

… …

0.2mL

0.2mL

Test serum

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

anticipated results1
Anticipated results

1.Hemolysis: clear, red

No hemolysis: turbid, ruddy

Hemolysis

No hemolysis

2.

attentions1
Attentions
  • Shake SRBC before use
  • Pipettes for different reagents should not be confused