Immunological assays, immunological assay & Bio luminescence

1. Immunological assays,
fluroimmuno assay &
Bioluminiscence
P Sohail
M Pharmacy, 1st year
Dept of Analysis

2. Outline
• Introduction
• Types of assays
• Immunological assays and techniques
• Bioluminiscence
• Conclusion

3. Assay
• An assay is an investigative procedure for qualitatively
assessing or quantitatively measuring the presence or
amount or the functional activity of a target entity (the
analyte) which can be a drug or biochemical substance or
organic sample

4. Types of Assays
• Chemical assays
• Immunoassays
• Microbiological assays
• Bioassay

5. Immunoassa
y

6. An immunoassay is a test that uses antibody and antigen
complexes as a means of generating measurable result
or
An immunoassay is an analytical method which uses
antibodies as reagents to quantitate or detect specific
analytes

7. Prerequisite
s
• ANTIGEN
• ANTIBODY
• ANALYTE
• LABEL

8. Principl
e• Immunoassay uses antibody and antigen complexes as a
means of generating measurable result

9. Types
• Competitive immunoassays.
• Non-competitive immunoassays.

10. Competitive Format
• In competitive formats, unlabelled analyte (usually antigen)
in the test sample is measured by its ability to compete
with labeled antigen in the immunoassay.
• The unlabeled antigen blocks the ability of the labeled
antigen to bind because that binding site on the antibody is
already occupied.

11. One step competitive
format
• Both the labeled antigen reagent (Ag*) and the unlabeled specimen
(or test sample analyte) compete for a limited amount of antibody.

12. Two step competitive
format• The antibody concentration of the reaction solution is present in
excess in comparison to the concentration of antigen
• Antibody reagent is first incubated with specimen containing
antigens of interest; then in the second step, labeled antigen is
added
• Improved assay sensitivity compared to one step assay formats

14. Noncompetitive (Sandwich) Method
• “Sandwich” assay because analyte is bound (sandwiched)
between two highly specific antibody reagents
• Provides highest level of assay sensitivity and specificity
• Applied to the measurement of critical analytes such as cardiac
and hepatitis markers

16. Homogen
eou
s
and
Het
ero
gen
eou
s
Im
mu
• Immunoassay methods that require separation of bound Ab-Ag*
complex are referred to as heterogeneous immunoassays. Those that
do not require separation are referred to as homogeneous
immunoassays.
• Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and therapeutic drugs.

18. Types
• ELISA
• Radioimmunoassay
• Fluoroimmunoassay

19. • Fluorescent compound in labeling the antibodies and antigen
• Europium fluoresces when it excite by light in the presence of a
developing solution
• It is 10 times more sensitive than RIA
FLUROIMMUNOASSAY

20. • A modern fluorescent based immunoassay uses as the
detection reagent a fluorescent compound which
absorbs light or energy (excitation energy) at a specific
wavelength and then emits light or energy at a different
wavelength
• The difference between the wavelength of the excitation
light and the emission light is called the Stokes shift.

21. Application
of
im
mu
noa
ssa
y
in
• Many of the macromolecule that can found in food are
good antigen and antibodies are capable of recognizing
them and small molecule.
• Composition of raw material and final product
• Harmful and useful minor substances with biological
activity (toxins and allergens)
• Enzyme detection
• Contaminants detection and determination (hormones
,drug, pesticides residue)

22. BIOLUMINISCENCE

23.  It is the production andemission of light by
alivingorganism.
 Widespread acrossmarine zooplankton and
micro-nektonic life.
(BIOS - LIVING,LUMEN -LIGHT)
BIOLUMINISCENCE

24.  Product of areaction
- Chemical (luciferin)
- Enzyme (luciferase).
Luciferin-indole derivative consisting of tryptamine,
arginine, and isoleusine.
MECHANISM

25. Luciferin + Luciferase Oxyluciferin,
(emitting light).

26.  “One of the most important processes in the
ocean, and yet hardly anyone wasstudying it.”
 Theseare absent in freshwater forms.
 Thesemostly the characteristic feature of
midwater and bottom dwelling deep seafishes.
BIOLUMINISCENCE IN FISH

27.  Fisheswith luminiscent organs are world wide
in distribution.
 Majority ofthem are bathy pelagic living at a
medium depth(500-2000).
 In fishes the luminescence is generally blueor
green.

28.  70%of all species
collected
from Bermuda and south
atlantic had light organs.
 Systematic survey shows 10-
15%of all marine fish
genera contain luminous
organs.

29.  New speciesof luminescent fish arediscovered
yearly
 But the function and physiology of the lightorgans
remain more speculation than scientificfact.
 Accessdifficulties, expensive ship and submarine
cost ,and low funding have hamstrung effect and
• our knowledge of bioluminescent fish lagsbehind.
CONCLUSION

30. 1. http://www.academia.edu/Documents/in/Bioluminescence
2. https://academic.oup.com/bioscience/article/69/7/487/5512573
3. https://en.m.wikipedia.org/wiki/Bioluminescence
4. https://en.m.wikipedia.org/wiki/Immunoassay
References:

31. THANK YOU