2. Lecturer :Prof. Dr. Abdullahi Sh. Hussein M.B.B.S, MD,
PhD
Professor of Internal Medicine and Immunology
&
Assistant Lecturer :
Dr. Mohamed Abdirahman Omar(Dr. Qalbi) MBChB
Faculty of Medicine and Surgery
Benadir University
www.benadiruniversity.net
3.
4.
5. Introduction:
๏ Immunofluorescence is the labeling of antibodies or
antigens with fluorescent dyes.
๏ This technique is sometimes used to make viral plaques
more readily visible to the human eye.
๏ Immunofluorescent labeled tissue sections are studied
using a fluorescence microscope.
๏ Fluorescein is a dye which emits greenish fluorescence
under UV light. It can be tagged to immunoglobulin
molecules.
6. ๏ There are two ways of doing IF staining
๏ Direct immunofluorescence
๏ Indirect immunofluorescence
1. Direct immunofluorescence
๏ Ag is fixed on the slide
๏ Fluorescein labeled Abโs are layered over it
๏ Slide is washed to remove unattached Abโs
๏ Examined under UV light in an fluorescent microscope
๏ The site where the Ab attaches to its specific Ag will show
apple green fluorescence
๏ Use: Direct detection of Pathogens or their Agโs in tissues or
in pathological samples
8. 2. Indirect immunofluorescence:
๏ Indirect test is a double-layer technique
๏ The unlabelled antibody is applied directly to the
tissue substrate
๏ Treated with a fluorochrome-conjugated anti-
immunoglobulin serum
9. ๏Advantage over direct IF
๏ Because several fluorescent anti-immunoglobulins can
bind to each antibody present in the first layer, the
fluorescence is brighter than the direct test.
๏ It is also more time-efficient since it is only one signal
labelled reagent, the anti-immunoglobulin, is prepared
during the lengthy conjugation process
20. ELISA is an immunologic assay modified in order
to determine solid phase bound proteins. The
assay is based on the interactions between antigen
(protein) and specific antibodies.
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
26. An important refinementโฆ. 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).
27. An important refinementโฆ. ELISA
๏ฎ The ELISA is a sensitive assay that is simple,
versatile, and reliable.
๏ฎ The ELISA is a particularly sensitive assay
because the detection and amplification is
enzyme dependent.
28. An important refinementโฆ. ELISA
๏ฎ Possible enzymes used in the ELISA system are
horseradish peroxidase (HRP) and alkaline phosphatase
(AP). Each has a high rapid conversion of chromogenic
substrate to coloured product resulting in high sensitivity.
๏ฎ In addition, the ELISA results can be measured
quantitatively by means of a spectrophotometer allowing
quick clinical analysis.
29. Principle of sandwich ELISA
๏ฎ First, the goat anti-human IgG is
adsorbed to the microwells plate.
The plates are washed to remove
excess unbound antibody.
Cognate antigen (IgG) is added
and binds to antibody.
wash
30. Principle of sandwich ELISA
๏ฎ Excess unbound antigen is washed off and HRP-
conjugated antibody (2st antibody) is added binding
still exposed determinants on the antigen. Excess
unbound 2st antibody
is washed away, and
chromogenic substrate added.
31. Principle of sandwich ELISA
๏ฎ Colour 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.
32. Protocol
1. The microwell plates have been coated with goat anti-
human IgG ( the 1st antibody) prior to the start of the
class lab.
Class lab starts with step 2.
2. Invert the microwell plate and dump the liquid out of
the wells.
33. 3. a) Place 100ยตl PBS-Tween in well No. 1 through No. 7 well.
b) Fill No. 1 well with 100ยตl original standard IgG.
c) Serial dilutions: Transfer 100ยตl from No. 1 well to No. 2 well ( 2-fold dilution). Mix,
repeat 2-fold dilution until come to No.7 well. Discard the 100ยตl extra from the last
well. You should end with 100ยตl in each well when you are finished.
d) Fill No. 8 well with 100ยตl sample , No. 9 well with 100ยตl PBS-T.
100ul PBS-T20
sample
100ul standard
100ul 100ul PBS control
blank
control
34. Procedure
4. Incubate the plate in a moisture container for 30 min
at 37โ. Wash the wells with PBS-Tween for 3
times.
โข To all the wells , add 100ยตl anti-human IgG
conjugated with HRP(1:5000 dilution in PBS-
Tween-BSA). incubate at 37 โ for 30 min.
โข Wash the wells as described in step 4.
35. Procedure
7. Add 100ยตl substrate to each well including blank,
sample or dilution. Do not touch the well or well
solution with tip, just drop in the 100ยตl substrate.
DO NOT create bubbles. The ELISA reader will
give false results if bubbles are present.
8. Incubate the plate for 30 min at 37 โ.
36. Procedure
10. Read absorbance of each well in microwell
spectrophotometer Reader. The absorbance
reading in No.1 well is the blank. single
wavelength at 405 nm. This absorbance value has
already been subtracted from all your samples.
From the standard curve determine concentration
(dilution) of unknown sample.
