1. Immunolabeling Technique

2. Immunnolabeling Technique
Definition: Immunnolabeling technique is methodology used for the quantitation of the specific antibody-antigen interaction by the attachment of a signal molecule to antibody and/or antigen. The signal molecules include enzymes , fluorochromes and radioisotopes.
Feature:
* combine both high sensitivity and high specificity of antigen-antibody reactions
* does not change the specificities of antigen-antibody reaction nor change the characteristics of the antigen and/or antibody.
Classification:
enzyme-linked immunosorbent assay ( ELISA) immunofluorescence
radioimmunoassay.

4. 1. Immunofluorescence
It is a method for localizing an antigen by use of fluorescently labeled antibodies.
One fluorescent compound that is widely used in immunology is fluorescein isothiocyanate (FITC), which fluoresces with a visible greenish color when excited by ultraviolet light. Another widely used fluorescent compound is phycoerythrin (PE), which fluoresces red.
It needs fluorescence microscope or spectrofluorometer

5. 2. Radioimmunoassay
Radioimmunoassay employs isotopically labeled molecules and permits measurements of extremely small amounts of antigen, antibody, or antigen-antibody complexes. The concentration of such labeled molecules is determined by measuring their ratioactivity, rather than by chemical analysis.
It needs special equipment such as radioactive counter.

6. 3. Enzyme-linked Immunosorbent Assay
(ELISA)
Definition
ELISA belongs to solid phase immunoassays, it employs the property of various plastics to adsorb monomolecular layers of proteins onto the surface. The presence of these proteins, bound to corresponding antigen or antibody adsorbed onto the plastic plate, may be detected by the use of anti- Ig, which is labeled with an enzyme.

7. The Feature of ELISA
The fundamental feature of the ELISA is to have an enzyme linked to an antibody and to have that enzyme be able to generate some visible product.
ELISA procedure may also be referred to as the EIA system (enzyme immunoassay).

8. The Feature of ELISA
The ELISA is a sensitive assay that is simple, versatile, and reliable.
The ELISA is a particularly sensitive assay because the detection and amplification is enzyme dependent.

9. The Feature of ELISA
Possible enzymes used in the ELISA system are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Each has a rapid conversion of chromogenic substrate to coloured product resulting in high sensitivity.
In addition, the ELISA results can be measured quantitatively by means of a spectrophotometer allowing quick clinical analysis.

10. The Feature of ELISA Application:
Antigens such as tumor markers, hormones and serum proteins may be determined by ELISA methodology
Classification:
indirect ELISA , sandwich ELISA competive ELISA

11. Indirect ELISA
The principle of indirect enzyme immunoassay was shown by the picture.. First, the antigen is adsorbed to an immobile surface such as the wells of a microtiter plate. The excess unbound antigen is washed off. Dilutions of test antibody is added to each immobilized antigen sample and allowed to bind to the antigen. The unbound antibody is washed away. This is followed by addition of a secondary antibody which is directed against the test antibody. The secondary antibody is conjugated to an enzyme. This enzyme catalyses a colourless substrate to color products. Again, the excess of this secondary conjugated antibody is washed away. Chromogenic substrate is added and colour develops which is directly proportional to the amount of bound antibody.

12. Competitive ELISA

13. Sandwich ELISA

14. 1. Immunnolabeling Technique
2. Measurement of IgG Level in Serum By Sandwich ELISA

15. Measurement of IgG Level in Serum By Sandwich ELISA

16. Materials standard IgG（for serial dilutions） Dilution solution(稀释液)
sample (样本)
HRP-conjugated antibody (the 2st antibody)
Blank solution(blank control)
PBS solution (PBS control)
PBS-Tween solution (洗涤液)
microwells plate (酶标反应板)
goat anti-human IgG ( the 1st antibody)
chromogenic substrate ：OPD-H2O2

17. Procedure
The microwells plates are already coated with goat anti-human IgG ( the 1st antibody), they were coated yesterday and kept at 4 ℃ overlight.
Class test starts with step 2.
2. Invert the microwells plate and pure the liquid out of the wells.
3. Wash the wells with PBS-Tween solution for three times. Fill the wells with PBS-Tween solution with pipette, every time allow the plate to stand for 30 sec and then pure PBS-Tween. Repeat this step twice.

18. 4. 1) Add 100µl PBS-Tween in well No. 1 to No. 7
2) Fill well No. 1 with 100µl original standard IgG.
3) Serial dilutions: Transfer 100µl from well No. 1 to No. 2 ( 2-fold dilution). Mix well and repeat 2-fold dilution up to No.7, Then discard the extra 100µl from No.7, You should end with 100µl in each well when you finished.
4) Fill well No. 8 with 100µl sample, No. 9 with 100µl PBS solution, No. 10 with 100µl blank solution.
100ul standard
100ul
100ul
PBS control
sample
blank control
100ul PBS-T20

19. Procedure
Put the plate into a moist container and incubate for 30 min at 37℃. Wash the wells as described in step 2 and 3.
To each well, add 100µl anti-human IgG conjugated with HRP(1:5000 dilution in PBS-Tween-BSA), incubate it at 37 ℃ for 30 min.
Wash the wells as described in step 2 and 3.

20. Procedure
8. Add 100µl substrate to each well. Do not touch the solution with tip, just drop in the 100µl substrate. Avoid to produce bubbles, because the ELISA reader will give false results if bubbles are present.
This substrate is chromogenic.
9. Incubate the plate for about 15min at room temperature.

21. Procedure
10. Read absorbance (OD value) of each well in spectrophotometer, at a single wavelength of 405 nm.
11. Draw a standard curve with the dilution of standard IgG for X axis, and the corresponding OD values as the Y axis, then from the standard curve determine the concentration of the sample.

22. Principle
First, the goat anti-human IgG is adsorbed to the microwells plate. The plates are washed to remove excess unbound antibody.
Corresponding antigen (IgG) is added and binds to antibody.
wash

23. Principle
Excess unbound antigen is washed off and HRP-conjugated antibody (2st antibody) is added to bind the antigen. Excess unbound 2st antibody
is washed away, and
chromogenic substrate added.

24. Principle
Color develops in wells containing the bound antigen. The absorbance of the solution in each well is proportional to the amount of bound antigen in it.