Immunofluorescence is a technique that uses the binding of antibodies to antigens to detect the presence of substances. It relies on tagging antibodies with fluorescent dyes called fluorophores. When exposed to ultraviolet light, the fluorophore-tagged antibody complexes emit visible light that can be seen using a fluorescent microscope. There are several types of immunofluorescence assays including direct, indirect, and quantitative assays that are used to detect microorganisms, antibodies, and other biomolecules through the antigen-antibody reaction and fluorescent signal produced.
Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC).
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
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.
Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC).
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
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.
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.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
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.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
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This presentation covers materials, common variations and necessary controls in a immunofluorescent staining protocol and a simple guide for troubleshooting.
Immunofluorescence or Immunofluorescence Antibody Assay(IFA) is a traditional laboratory technique that utilizes fluorescent dyes to identify the presence of antibodies bound to specific antigens.
Immunofluorescence is a powerful technique which allows for the visualisation of proteins or antigens within a cell or a section of tissue. Utilizing the binding specificity of an antibody to a given antigen, antibodies chemically conjugated with fluorescent dyes can be used to bind to specific molecular targets. This allows for easy visualisation by confocal or fluorescent microscopy.
Immunoprecipitation: Procedure, Analysis and Applicationsajithnandanam
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
Pharmaceutical biotechnology ..in that different blotting techniques such as ELISA , western blotting and southern blotting.There applications and their advantage and disadvantage with their diagrams
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.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
2. WHAT IS IMMUNOFLUORESCENCE?
• A qualitative observation; uses a fluorescent microscope
• Relies on the principle of antigen (Ag)-antibody (Ab) reaction:
> Ab is “tagged” with a fluorophore or fluorochrome (fluorescent
compound)
> Ab binds to a specific Ag of interest
>Ag-Ab complex emits a green or red light (depending on the
fluorophore used) when an incident light (usually UV light) is introduced
• Introduced by Albert Coons in 1941
3. WHAT IS A FLUOROPHORE
• Organic molecules with a ring structure
• Has a characteristic optimum absorption range
(e.g. Fluorescein isothiocyanate emits a GREEN light when against a BLUE light)
• ABSORBS incident light, CONVERTS it, and EMITS fluorescent energy of longer
wavelength and lower energy
4. EXAMPLES (FLUOROPHORES)
• Fluorescein isothiocyanate – most common; emits green light
• Tetramethylrhodamine – emits red light
• Phycoerythrin
• Europium (β – naphthyl trifluoroacetone)
• Lucifer Yellow VS
• Acridine Orange
• Lissamine
• Calcofluor White
5. WORKING PRINCIPLE
• Employs Ag-Ab reaction
• Ab tagged with a fluorophore is introduced to specimen
• Ab binds to specific Ag
• Specimen is viewed under a UV light with a dark background in a fluorescent
microscope
6. WORKING PRINCIPLE (CONT.)
• Fluorophore absorbs radiant energy and is excited
• Fluorophore goes back to a ground state, emitting light energy of longer
wavelength and lower energy
• Interval of absorption and emission is very short; occurs in nanoseconds
7. PURPOSE OF
IMMUNOFLUORESCENT ASSAYS
• Rapid identification of microorganisms in cell culture or infected tissue, tumor-
specific Ag on neoplastic tissue, transplantation Ag, and CD Ag on T and B cells (via
Cell Flow Cytometry)
8.
9.
10. TYPES OF
IMMUNOFLUORESCENT ASSAYS
1. Direct Immunofluorescence Assay (DIFA)
Tagged Ab is directly added to an unknown Ag fixed to a slide
Requires incubation and a wash step
Ag are seen as bright apple green or orange-yellow against a dark backgrounds
Suitable for detection of specific Ag in tissue or body fluids
e.g. Legionella pneumophila, Pneumocystis carinii, Chlamydia
trachomatis, respiratory syncytial virus (RSV)
11. TYPES OF
IMMUNOFLUORESCENT ASSAYS
2. Indirect Immunofluorescent Assay
Specimen with a known Ag is incubated on a solid phase
Antihuman immunoglobulin (from mouse) tagged with a fluorophore is added
Specimen Ab binds with antihuman Ab, forming a sandwich and localizing
fluorescence
Used in Ab identification and detection of Treponema species-specific, antinuclear,
chlamydial, and toxoplasma Ab, and Ab to herpes simplex virus (HSV), Epstein-Barr
virus (EBV), and Cytomegalovirus (CMV)
12. TYPES OF
IMMUNOFLUORESCENT ASSAYS
ADVANTAGE (over DIFA):
a.) Uses only one Ab conjugate (tagged Ab) for different reactions, eliminating the
need for numerous purified, labeled reagent antibodies)
b.) More sensitive; has increased staining property. Multiple molecules bind to each
primary molecule.
13. TYPES OF
IMMUNOFLUORESCENT ASSAYS
3. Microimmunofluorescence
Detects Ab in patient serum
Has the same working principle as Indirect Immunofluorescence Assay but employs
Teflon slides with many wells dotted with Ag
Used for:
a. Serodiagnosis of Q fever, Mediterranean Spotted Fever
b. Detection of IgG, IgA, and IgM Ab to Chlamydia, Toxoplasmosis, epidemic
Typhus, etc.
14. TYPES OF
IMMUNOFLUORESCENT ASSAYS
4. Quantitative Fluorescent Immunoassays (FIAs)
can be classified as heterogenous or homogenous, depending on the type of
enzymatic immunoassays
label is fluorescent; can be applied to either Ag or Ab
Solid-phase heterogenous fluorescent assay is employed for the identification of
Ab to nuclear Ag, Toxoplasma Ag, Rubella Ag, and numerous other virus Ag
Can also be used to detect important biological compounds
e.g. Cortisol, Progesterone, serum Thyroxine (T4)
15. TYPES OF
IMMUNOFLUORESCENT ASSAYS
Homogenous FIA require no separation procedure; rapid and simple
• Has only one incubation step and no wash step
• Competitive binding is involved
• Fluorescent label changes as the conjugated Ag binds to specific Ab
• Changes include wavelength emission, rotation freedom, polarity, or dielectric strength
• Amount of fluorescence is proportional to the amount of Ag present; as Ag binding
increases, binding of fluorescent analyte decreases, which gives off more fluorescence
16. TYPES OF
IMMUNOFLUORESCENT ASSAYS
Fluorescence Polarization Immunoassay (FPIA) – based on the change in
polarization of fluorescent light emitted by the fluorophore of a tagged Ab
• Incident light directed to a specimen is polarized with a lens or a prism; waves are
aligned at one plane
• Labelled molecules bound to an Ab rotates less and emits an increased amount of
polarized light
• Degree of fluorescence of polarization is INVERSELY PROPORTIONAL to concentration
of the analyte
• Limited to small molecules that tumble freely in the solution (< 2000 Daltons)
• Nonspecific binding of tagged Ab to other serum proteins occur, increasing polarization,
and falsely decreasing values
17. TYPES OF
IMMUNOFLUORESCENT ASSAYS
• Used mainly in determining therapeutic drug concentrations and hormone
concentrations
• Requires sophisticated instrumentations; basis for several automated analyzers
18.
19. ADVANTAGE AND DISADVANTAGE OF
FLUORESCENT IMMUNOASSAY
ADVANTAGES DISADVANTAGES
• Has the potential of being highly sensitive and
versatile
• Separation of signal on the tag from the
autofluorescence produced by different
substance in serum
• Methodology is fairly simple • Nonspecific binding causes quenching;
fluorescence generated is changed
• No need to deal with or dispose of hazardous
substances
20. ADVANTAGE AND DISADVANTAGE OF
FLUORESCENT IMMUNOASSAY
• FPIA was introduced to overcome some of the problems
• Requires expensive dedicated instrumentation; limits its use in smaller laboratories
21. REFERENCES:
• Stevens, C. D.. (2010). Clinical Immunology & Serology in Laboratory Medicine, (3rd
ed.). Philadelphia, PA: F.A. Davis Company
• Sridar, R.P.N.. (2006). Immunofluorescence. Retrieved April 11, 2016, from
http://www.microrao.com/micronotes/immunofluorescence.pdf
• Abcam. (n.d.). Direct vs indirect immunofluorescence. Retrieved April 11, 2016, from
http://www.abcam.com/secondary-antibodies/direct-vs-indirect-
immunofluorescence
