The document provides an introduction to ELISA (enzyme-linked immunosorbent assay), which is a biochemical technique used mainly in immunology to detect the presence of an antibody or antigen in a sample. It describes the basic principles and steps of the ELISA process, which involves detecting antibodies or antigens using an enzyme-labeled secondary antibody and color changing reaction. Key aspects covered include antigen-antibody binding, use of enzyme labels, substrate conversion, and quantitative/qualitative applications of ELISA for detecting various molecules.
ELISA- Principle, procedure , types and applicationsJaskiranKaur72
Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays.
This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones.
The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result.
ELISA- Principle, procedure , types and applicationsJaskiranKaur72
Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays.
This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones.
The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
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.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
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.
Laboratory method for measuring enzyme activity.
Vital for study of enzyme kinetics and enzyme inhibition.
Measurement of enzyme activity – follow the change in concentration of substrate or product – measure reaction rate.
ELISA use an enzyme to detect the binding of antigen (Ag) antibody (Ab). • The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. • An ELISA can be used to detect either the presence of antigens or antibodies in a sample depending how the test is designed
ELISA or Enzyme-linked Immunosorbent Assay is a qualitative and quantitative assay for detecting the presence of antigens (virus, hormones, enzymes, etc.) in a sample.
Enzyme linked immunosorbent assay (elisa) and its clinical significancerohini sane
A comprehensive presentation on Enzyme Linked Immunosorbent Assay (ELISA) and its clinical significance for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.
Test for detection of plant virus by ELISA test.pdfLOKESH R
This presentation provides an overview of the Enzyme-Linked Immunosorbent Assay (ELISA) test for the detection of plant viruses. The ELISA test is a highly sensitive and specific method for detecting viral antigens in plant tissues, seeds, and other plant materials.
The presentation covers the principles of ELISA test, including the different types of ELISA tests, such as direct, indirect, sandwich, and competitive ELISA. The steps involved in the ELISA test, including sample preparation, antibody labeling, incubation, and detection, are discussed in detail.
The advantages and limitations of the ELISA test for plant virus detection are also highlighted. The presentation includes a comparison of the ELISA test with other diagnostic methods for plant viruses, such as polymerase chain reaction (PCR) and serological assays.
Finally, the presentation provides examples of the applications of the ELISA test for plant virus detection, including its use in crop protection, disease management, and quarantine measures. The presentation concludes with a summary of the key points discussed and recommendations for the use of the ELISA test in plant virus detection.
This lecture is presented by our volunteer M. Fida ur Rehman, he is from Pakistan, and he is covering ELISA topic.
For video: https://www.youtube.com/watch?v=ZEEgRHUIbJ0
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www.seribangash.com
A Memorandum of Association (MOA) is a legal document that outlines the fundamental principles and objectives upon which a company operates. It serves as the company's charter or constitution and defines the scope of its activities. Here's a detailed note on the MOA:
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Name Clause: This clause states the name of the company, which should end with words like "Limited" or "Ltd." for a public limited company and "Private Limited" or "Pvt. Ltd." for a private limited company.
https://seribangash.com/article-of-association-is-legal-doc-of-company/
Registered Office Clause: It specifies the location where the company's registered office is situated. This office is where all official communications and notices are sent.
Objective Clause: This clause delineates the main objectives for which the company is formed. It's important to define these objectives clearly, as the company cannot undertake activities beyond those mentioned in this clause.
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https://seribangash.com/promotors-is-person-conceived-formation-company/
Capital Clause: This clause specifies the authorized capital of the company, i.e., the maximum amount of share capital the company is authorized to issue. It also mentions the division of this capital into shares and their respective nominal value.
Association Clause: It simply states that the subscribers wish to form a company and agree to become members of it, in accordance with the terms of the MOA.
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Legal Requirement: The MOA is a legal requirement for the formation of a company. It must be filed with the Registrar of Companies during the incorporation process.
Constitutional Document: It serves as the company's constitutional document, defining its scope, powers, and limitations.
Protection of Members: It protects the interests of the company's members by clearly defining the objectives and limiting their liability.
External Communication: It provides clarity to external parties, such as investors, creditors, and regulatory authorities, regarding the company's objectives and powers.
https://seribangash.com/difference-public-and-private-company-law/
Binding Authority: The company and its members are bound by the provisions of the MOA. Any action taken beyond its scope may be considered ultra vires (beyond the powers) of the company and therefore void.
Amendment of MOA:
While the MOA lays down the company's fundamental principles, it is not entirely immutable. It can be amended, but only under specific circumstances and in compliance with legal procedures. Amendments typically require shareholder
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3. INTRODUCTION TO ELISA
ELISA, or enzyme-linked immunosorbent assay,
are quantitative immunological procedures in
which the Ag- Ab reaction is monitored by
enzyme measurements.
The term ELISA was first used by Engvall &
Perlma in 1971.
The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.
5. ELISA is the abbreviation of
ENZYME-LINKED
IMMUNOSORBENT ASSAY
It is useful & powerful
method in estimating ng/mL
to pg/mL ordered materials
in the solution .
6. Why known as ......?
Enzyme Linked Immunosorbent Assay
1. Antigen of interest is absorbed on to plastic surface
(„sorbent‟).
2. Antigen is recognised by specific antibody („immuno‟).
3. This antibody is recognised by second antibody
(„immuno‟) which has enzyme attached („enzyme-
linked‟).
4. Substrate reacts with enzyme to produce product, usually
coloured.
7. BASIC PRINCIPLE OF ELISA
Use an enzyme to detect the binding of antigen (Ag)
antibody (Ab).
The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating the
presence of Ag : Ab binding.
An ELISA can be used to detect either the presence
of Antigens or antibodies in a sample depending how
the test is designed.
ELISA was dveloped in 1970 and became rapidly
accepted
8. Secondary
antibody
Substrate
Coloured
product
Primary
antibody
Different antigens in sample
9. ELISA Qualitative/Quantitative
Qualitative
determines antigen or antibody is present or absent
Quantitative
determines the quantity of the antibody
Titer
The highest dilution of the specimen usually serum
which gives a positive reaction in the test
11. ANTIGEN (Ag)
Any molecule that induces production of antibodies
when introduced in the body of an animal is called
antigen.
OR
any “thing”, foreign to the immune system. e.g.
bacteria, viruses, (or their parts), pollen, etc.
Protein molecule SYMBOL FOR ANTIGEN
Carbohydrate molecule.
Microorganisms
Allergens.
Viruses Etc.
12. ANTIBODY ( Ab)
Antibody: proteins produced by the immune
system which help defend against antigens
SYMBOL FOR
ANTIBODY
Y
21. Competitive Elisa
Used to determine small molecule antigens.(T3,T4,progesterone etc.)
antibody coated microwell
serum antigen and labelled antigen added together--competition.
antibody-antigen-enzyme complex bound is inversely related to the
concentration of antigen present in the sample.
The bound enzyme conjugate reacts with the chromogenic substrate added to
produce a color reaction (blue to yellow color). .
Increased serum antigen results in reduced binding of the antigen-enzyme
conjugate with the capture antibody producing less enzyme activity and color
(yellow) formation
Substrate product concentration is inversely
proportional to the concentration of standard or test
antigen added
23. Noncompetitive
Sandwich Assay
Direct Assay
Antigen capture ELISA Antigen adsorbed directly detected by labeled
enzyme
Antibody capture ELISA Antibody adsorbed directly by labeled enzyme.
Indirect Assay
Antigen directly adsorbed onto the solid phase is first incubated with
patient serum, and then with a labeled antibody specific for human
immunoglobulin.
Detection of infectious agents(HIV,HBV,HCV) and auto antibodies
In Indirect ELISA color change is directly proportional
to the concentration of specific antibodies in specimen
24. Sandwich Assay
Antigens such as tumor markers, hormones and serum proteins may be
determined
Antigen in the sample binds with the capture antibody on the microwell
and becomes immobilized.
The antibody of the enzyme conjugate binds with the immobilized
antigen to form a sandwich of antibody-antigen-antibody/enzyme bound
to the microwell.
Enzyme reaction product is directly proportional to
concentration of standard or analytical antigen
26. to detect Ab (HIV, HCV)
to detect Ag ( Tumor Markers, Hormones )
to detect Ag ( Free Testosterone)
Comparison between Indirect Sandwich & Competitive ELISA
28. Importance of incubation step:-
During the test performance incubation time and
mentioned temperature is must required For the
proper binding between antigen and antibody and
also binding with conjugate and color development
of substrate.
Importance of Washing :- For the removal of any
unbound Antibody/Antigen proper washing and
taping is required other wise we get the incorrect
result.
So incubation & washing is much important for good
results.
29. Enzymes Used in Elisa
Horseradish peroxidase (most commonly used)
Alkaline Phosphatase
β-galactosidase
Lactoperoxidase
Tetra Methyl benzidine
In case of peroxidase, the substrate hydrogen peroxide is converted
into water and o2 in the presence of electron donors . (like
diaminobenzidine or 4-chloronaphthol which themselves oxidized in
the reaction).
Oxidation of diaminobenzidine produces dark brown color while that
of 4-chlorornaphthol yields purple color which is the basis of ELISA
30. ENZYME SUBSTRATE
Initially the substrate should be colorless
After degradation by the enzyme it should be
strongly colored or fluorescent.
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline p-NPP p-NPP+ 1 M NaOH
Phosphatase diethandamine+Mg
Cl2
Horse radish H2O2 Tetramethylbenzidi 1 M H2SO4
Peroxidase ne + Phosphate –
Citrate buffer
Horse radish H2O2 O– 1 M HCl
Peroxidase Phenylenediamine +
HCl
31. ELISA KIT FOR DETERMINATION OF IgA, IgG or
IgM anti-Mycobacteria antibodies in human serum
ANDA-TB DETECTION OF MYCOBACTERIA
In vitro diagnostic test for the determination of IgA, IgG and IgM antibodies against mycobacteria in
human liquid (serum, CSF, pleural fluid, sputum, saliva, etc...)
33. What Are The
Reagents?
Antigen: Elisa plate coated with the A60
And antigen-antibodies complexes.
What Function Primary antibody: Human Serum IgG, IgA, &
IgM
Do They
Secondary antibody: Peroxidase-labelled anti-
Perform? human IgA, IgG, or IgM antibodies that bind
to the antibobdy complexes .
Enzyme substrate: 3,3’,5,5’ –
tetramethylbenzidine (TMB) – a colorless
solution that when oxidized by HRP turns
blue.
Stop Solution: Sulphuric Acid 0.5N (H2So4)
34. Elisa Plate
Microtitre wells
Generally 96
wells
Marked on one
side alphabetically
Numerically on
the other side
Comes with the
kit
35. Complete forms and specimen
identification
Fill in the information on tube (identifying
information, date of collection, and other
information as required).
Fill in the laboratory form that will accompany
specimens.
For TB Gold used heparin Tube
FFFI
36. Collection and processing of
serum
Collect blood in a tube that does not contain any
chemicals or anticoagulants.
Collect 5mL of whole blood (for very small children
collect 1mL).
Place tube upright for 30-60 minutes then when firm
clot has formed, centrifuge tube for 20 minutes at
2500rpm.
Remove serum with a pipette and place in a plastic
storage tube (2-3mL microtube or cryovial).
If 5mL of blood was collected it will result in about
2mL of serum.
37. TEST PERFORMANCE
Using a clean
Pipette , add 100 µL
of diluted serum
sample (Dilute the
sera to be tested
1:100 in the sample
diluents) to each
well.
Incubate 1 hour at
37°C .
38. After incubation empty out contents of
wells into waste container.
Using pipette, fill wells with washing
buffer then empty out.
Tap wells upside down on paper towel.
Wash the wells 5 times. At the end of the
washing process, the wells must be
entirely dry after the last wash.
39. Distribute 100µL of
anti-human
immunoglobulin-POD
conjugate in each well.
Incubate 30 minutes at
37°C.
40.
41. ELISA PLATE READY
FOR READING
Measures the absorbance
at 450nm With the help of
ELISA READER.
Calculate the absorbance
for each sample and
reference.
We used Ascent Software
for Calculation of the
result
43. RESULT DETERMINATION:-
A) IgA and IgG Tests:-
Plot the O.D. result of each reference , except for the negative reference on the
vertical axis (Y-axis) in relation to the number of corresponding units on the
horizontal axis (X-axis).
Using the absorbance value for each sample , determine the corresponding
concentration of antibodies expressed in units/ml from the reference curve.
B) IgM Tests :-
We can calculate the cut off value A450nm sample / A450nm Positive limit
reference
the normalized value of the positive reference is 1 .
All samples whose value is comprised between 0.8 and 1.0 are considered
dubious and all samples whose normalized value is above 1.0 are considered
positive for IgM antibodies.
44. What is Cut-off Value ………?
Cut-off: provided in the kits by the manufacturer. The cut-off value
defines a range in which 90% of the normal population is negative below
the cut-off value and 10% of the normal population is positive above the
cut-off value.
ELISA is semiquantitative method. The calculation is done as
follows. The units of ELISA is OD ratio:
Sample value= sample OD/cut-off OD
46. Advantages of ELISA
Reagents are relatively cheap & have a long
shelf life
ELISA is highly specific and sensitive
No radiation hazards occur during labelling or
disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely
available.
ELISA can be used to a variety of infections.
47. Disadvantages of ELISA
Measurement of enzyme activity can be more complex
than measurement of activity of some type of
radioisotopes.
Enzyme activity may be affected by plasma
constituents.
Kits are commercially available, but not cheap
Very specific to a particular antigen. Won‟t recognize
any other antigen
False positives/negatives possible, especially with
mutated/altered antigen
48. Limitations
•Results may not be absolute
•Antibody must be available
•Concentration may be unclear
•False positive possible
•False negative possible
49. APPLICATIONS OF ELISA
1- Hormones 7- Vaccine Quality Control
2- Proteins 8- FOR GMO (Genetically modified
organism)
3- Infectious Agent ( Viral, Bacterial, 9- For Rapid Test
Parasitic, Fungal )
4- Drug Markers 10- IgG, IgM, IgA
5- Tumor Markers 11- In New Born Screening
6- Serum Proteins 12- In Clinical Research
51. A New technique:- Reverse ELISA
A new technique uses a solid phase made up of an immunosorbent
polystyrene rod with 4-12 protruding ogives. The entire device is immersed
in a test tube containing the collected sample and the following steps
(washing,incubation in conjugate and incubation in chromogenous) are
carried out by dipping the ogives in microwells of standard microplates pre-
filled with reagents.
Advantage of this technique:
1- The ogives can each be sensitized to a different reagent, allowing the
simultaneous detection of different antibodies and different antigens for
multi-target assays.
2- The sample volume can be increased to improve the test sensitivity in
clinical, food and environmental samples.
3- One ogive is left unsensitized to measure the non-specific reactions of the
sample.
4- The use of laboratory supplies for dispensing sample aliquots, washing
solution and reagents in microwells is not required, facilitating ready-to-use
lab kits and on-site kits.