Immunolabeling Technique. Immunnolabeling Technique.
* 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.
enzyme-linked immunosorbent assay ( ELISA) immunofluorescence
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.
Antigens such as tumor markers, hormones and serum proteins may be determined by ELISA methodology
indirect ELISA , sandwich ELISA competive ELISA
2. Measurement of IgG Level in Serum By Sandwich ELISA
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.
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.
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.
is washed away, and
chromogenic substrate added.