ELISA &

MICROSCO
PY

HAIDER ALI
IMMUNOLOGY & MOLECULAR
PATHOLOGY (70172292)
OBJECTIVES
 ELISA
 History of ELISA
 Types of ELISA
 Abuse Drug Testing by using ELISA
 Microscopy
 History of Microscope
 Types of Microscopes
INTRODUCTION TO ENZYME-LINKED
IMMUNOSORBENT ASSAY (ELISA)

 Elisa or Enzyme-linked immunosorbent assay is an immunoassay

technique involving antigen and antibody reaction in vitro.
 Elisa is a sensitive technique and specific assay for detecting and
quantitating antigens and antibodies.
 Elisa is a microwell plate-based technique, used for detecting and
quantifying Substances such as peptides, proteins, antibodies,
hormones, and Drugs.
HISTORY OF ELISA

 Radioimmunoassay was first described in a Scientific Paper by

Rosalyn Sussman and Solomon Berson, Published in 1960.
 However, Concerns regarding the potential health risks of
radioactivity prompted researchers to find a safer alternative.
 In 1971, Peter Perlman and Eva Engvall developed a
revolutionary method Called the ELISA.
 At Stockholm University Sweden.
PRINCIPLE OF ELSA

 Elisa is based on the binding of antibodies to specific

antigens, followed by enzymatic detection.
ELISA REQUIREMENTS
 Coated plates (Microtiter plates): Commonly used ones are 96 well
polystyrene plates. Coated with antigens or antibodies at the bottom of
the well.
 Sample diluents: To dilute the sample before application in some cases
of ELISA test.
 Wash Buffers: Help to wash away the unrequired contaminants and
unbound antigens or antibodies. E.g., Triphosphate buffer (pH 7.40) and
detergents such as Tween-20.
 Enzyme-linked Antibodies: For this, most common enzymes used are
• AP(Alkaline Phosphatase)
• HRP(Horseradish Peroxidase)
CONTINUE …

 Substrates: Specific chromogenic substrates are used for the

respective enzymes used. Commonly used substrates are o-
phenylene diamine for HRP enzyme and p-nitrophenyl phosphate for
AP enzyme.
 Stop solution: It stops the enzyme and substrate reaction. It can
be acids such as sulphuric acid.
ELISA KIT
 Calibrato
rs
ELISA
Microwell Plate ELISA
Washer Reader
GENERAL PROCEDURE
 Antigen or Antibody is immobilization on microplate.
 Sample addition (target Ag/Ab) and Incubation.
 Specific binding occurs between the immobilized molecule and the target
molecule
 Washing
 Enzyme-linked antibody addition, binding, and Incubation.
 Washing
 Chromogenic Substrate addition, converting to colored product and Incubation.
 Stop Solution addition.
 Absorbance Measurement, Proportional to the target molecule concentration.
TYPES OF ELISA

 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA
DIRECT ELISA
 Antigen Detection
 In Direct ELISA, an antigen or sample is immobilized directly on a
microplate and a conjugated detection antibody binds to the target
protein.
 Substrate is then added, Producing a signal proportional to the
amount of analyte in the sample.
DIRECT ELISA
INDIRECT ELISA
 Primary Antibody Detection
 Indirect ELISA is a two-step binding process involving primary and
labeled secondary antibodies.
 In this method, the primary antibody is incubated with the antigen-
coated wells.
 Next a labeled secondary antibody that recognizes a primary antibody
is added.
 Substrate is then added, Producing a signal proportional to the amount
of analyte in the sample.
INDIRECT ELISA
SANDWICH ELISA

 Antigen Detection
 A sandwich ELISA measures the Antigen between two layers of
antibodies (Capture and detection Antibodies)
 The target antigen must contain at least two antigenic sites capable
of binding to antibodies.
SANDWICH ELISA
COMPETITIVE ELISA
 Antigen or Antibody Detection
 For Antigen Detection, Antibody is first incubated with a sample containing
antigen.
 Then this mixture is then added to the microplate which is coated with antigen.
 The more the antigen present in the sample, the less the free antibody will be
available to bind to the antigen-coated well.
 Enzyme Conjugated Secondary antibody is then added.
 Substrate is then added.
 The Absorbance is inversely proportional to the Concentration of Ag in the
sample.
COMPETITIVE ELISA
 TYPE ADVANTAGES DISADVANTAGE
S
Direct ELISA • Quick Methodology since only one • Immune
antibody is used.
Reactivity
primary antibody may be
of

• Cross-Reactivity of secondary antibody reduced as a result of

is eliminated. labeling.
• Little signal Amplification.

Indirect ELISA • Immunoreactivity of Primary antibody • Cross Reactivity may occur

is not affected by labeling. with the secondary antibody,
• Sensitivity is increased because each resulting in the non-specific
Pri. Ab contains several epitopes for signal.
labeled Sec. Ab. • Extra Incubation step is
required.

Sandwich • Highly Sensitive and Specific as two • Complex

antibodies are used to detect the
workflow
Requires more incubation
–

ELISA antigen. steps.

• It offers flexibility since both direct and • Requires more optimization –
indirect methods can be used. cross-reactivity between
different antibodies must be
checked.
APPLICATION
 Screening donated blood for evidence of viral contamination by
 HIV-1 and HIV-2 (presence of anti-HIV antibodies)
 hepatitis C (presence of antibodies)
 hepatitis B (testing for both antibodies and a viral antigen)

 Measuring hormone levels

 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)

 TSH, T3 and T4 (for thyroid function)

APPLICATION
 Detecting Infections
 Sexually Transmitted agents like HIV, Syphilis, and Chlamydia
 Hepatitis B and C
 Herpes Virus
 TORCH Profile

 Detecting Allergens in food and house dust

 Detecting illicit drugs e.g.,
 Cocaine
 Opiates
DRUG ABUSE TESTING USING ELISA

 We can use the following types of ELISA for Drug Abuse Testing ;
 Direct ELISA
 Indirect ELISA
 Competitive ELISA
DRUGS OF ABUSE
 Marijuana
 Cocaine
 Opiates
 Amphetamines
 Benzodiazepines
 Barbiturates
PURPOSE OF DRUG ABUSE TESTING IN FORENSICS

 Criminal Investigation
 Toxicology Screening
 Death Investigation
 Sexual assault Cases
TYPES OF SAMPLES
 Urine (Detection Window 1 to 5 Days)
 Centrifuge 3000 rpm/5 min and the supernatant is used for testing

 Blood (Hours to Days)

 Centrifuge 3000 rpm/5 min and Serum/Plasma is used for testing

 Saliva (Hours to Days)

 Centrifuge 3000 rpm/5 min and Supernatant is used for testing

 Hair (Weeks to Month)

 Hair Collection (100-200) – Hair Segmentation (1-3 cm) – Washing with
methanol/Acetone – Dry (air dry/Oven dry) – Hair Digestion by using
enzymes/Acids – Extraction of drugs by using Methanol/Ethanol
COMPETITIVE ELISA (CELISA) FOR DRUG ABUSE
TESTING
 Microplate coated with antibody specific to the target drug.
 Sample containing the target drug and enzyme-labeled drug
conjugate added.
 Competition for antibody binding between and target drug and
conjugate.
 Conjugate binding is inversely proportional to target drug
concentration.
COMPETITIVE ELISA (CELISA) FOR DRUG ABUSE
TESTING
PRECAUTIONS
NEGATIVE CONTROL WITH STRONG SIGNALS

 Inadequate Rinsing of Plates

 Reagents, not sufficiently diluted
 Non-specific binding of Enzyme Conjugate
 Cross-reactivity of Sec. antibody with the components in the antigen
Sample
POSITIVE CONTROL WITH NO SIGNAL

 Microwell Plates not coated properly

 Reagents applied in the wrong order
 Enzyme conjugate defective or inhibited by contaminant
 Secondary antibody not matched with the species of the Primary
antibody
ELISA WITH WEAK SIGNAL

 Wash buffer not adequately drained after every wash step

 Inadequate incubation times
 Detection reagents too dilute
 Enzyme and substrate defective or inhibited by contaminant
 Microwell plates poorly coated
 CO
OS
R
IC
M
P Y
MICROSCOPY

 The Science of investigating small objects using an instrument

(Microscope) is Called Microscopy.
Microscope
 A Microscope (Greek: mikron = Small and Scopeos = To look)
 Is an Instrument for viewing Objects that are too small to be seen by
naked or unaided eye.
HISTORY OF MICROSCOPE

 1590 – Hans Janssen and his son

Zacharias Janssen, Developed
the first microscope. Janssen’s
Microscope
 1609 – Galileo – Occhiolino or
Compound Microscope
HISTORY OF MICROSCOPE

 1620 – Christian Huygens,

developed a simple two-lens
Ocular System.
 Anton Van Leeuwenhoek (1632-
1723)
 Robert Hooke (1635-1703)
PARTS OF MICROSCOPE
STRUCTURAL PARTS OF MICROSCOPE

 There are Three Structural parts of the Microscope:

 Head: Is also known as the body, it carries the optical parts in the upper
part of the microscope.
 Base: It acts as microscope support. It also carries microscopic
illuminators.
 Arms: This is the part connecting the base to the head. It supports the
head of the microscope and is also used when carrying the microscope.
OPTICAL PARTS OF MICROSCOPE

 The Optical parts of the microscope are used to view, magnify, and
produce an image from a specimen placed on a slide. These Parts
Include:
 Eye Piece: Also known as the ocular, this is the part used to look through the
microscope. It is found at the top of the microscope. Its Standard magnification is
10x with an optical eyepiece having magnifications from 5x to 10x
 Objective Lenses: These are the major lenses used for specimen visualization.
They have a magnification power of 40x to 100x. One microscope has 1 to 4
objective lenses, each with its own magnification power.
OTHER PARTS
 Nose Piece: also known as the revolving turret. It holds the objective
lenses. It is movable hence it can revolve the objective lenses depending
on the magnification power of the lens.
 Adjustment Knobs: These are knobs that are used to focus the
microscope. There are two types of adjustment knobs i.e. fine adjustment
knobs and coarse adjustment knobs.
 Stage: This is the section where the specimen is placed for viewing. They
have stage clips that hold the specimen slides in place.
 Aperture: This is a hole in the microscope stage, through which the
transmitted light from the source reaches the stage.
CONTINUE …

 Diaphragm: it’s also known as the iris. It is found under the stage of the
microscope and its primary role is to control the amount of light that
reaches the specimen.
 Microscopic Illuminator: This is the microscope’s light source, located at
the base. It is used instead of a mirror. it captures light from an external
source of a low voltage of about 100v.
 Condenser: These are lenses that are used to collect and focus light from
the illuminator into the specimen. They are found under the stage next to
the diaphragm of the microscope.
PARAMETERS OF MICROSCOPE

 There are two Parameters of Microscopy:

 Magnification: Measures how much larger a microscope causes an object to
appear.
Total Magnification = Magnifying Power of Objective lens X Magnifying Power of
Ocular lens
400x = 40x X 10x
 Resolving Power: the resolution of a microscope or lens is the smallest
distance by which two points can be separated and still be distinguished as
separate objects.
TYPES OF MICROSCOPE
1) SIMPLE MICROSCOPE

 A simple microscope uses a single lens for magnification such as a

convex lens of small focal lengths.
 A simple microscope works on the principle that when a tiny object
is placed within its focus, a virtual, erect, and magnified image of
the object is formed at the least distance of distinct vision from the
eye held close to the lens.
SIMPLE MICROSCOPE

 Uses of Simple Microscope

 It is typically used to examine minute
algae, fungi, and biological materials
 Watchmakers frequently use it to obtain a
magnified image of minor elements of a
watch.
2) COMPOUND MICROSCOPE
 The term compound in the Compound microscope refers to the microscope having
more than one lens (Objective Lens and Ocular Lens).
 It comes with its own Light source.
 The specimen or object, to be examined is usually mounted on a transparent glass
slide and positioned on the specimen stage between the condenser lens and
objective lens.
 A beam of visible light from the base is focused by a condenser lens onto the
specimen.
 The objective lens picks up the light transmitted by the specimen and creates a
magnified image of the specimen called the primary image inside the body tube.
This image is again magnified by the ocular lens or eyepiece.
COMPOUND MICROSCOPE

 Uses of Compound Microscope

 A compound microscope is of great use
in pathology labs to identify diseases.
 In forensic labs, it is used to identify
the presence of minerals or metals in
human cells to solve criminal cases.
 Plant cells and also the microbes living
on them can be observed under it
3) CONFOCAL MICROSCOPE

 Confocal microscopy, most frequently confocal laser scanning

microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an
optical imaging technique for increasing optical resolution and contrast
of a micrograph by means of using a spatial pinhole to block out-of-focus
light in image formation.
 Utilizing state-of-the-art technology and lasers that separate light waves,
you can view images without blurred edges and in higher resolutions.
CONFOCAL MICROSCOPE

 Uses of Confocal Microscope

 Study of Biofilms
 can form on many different types
of surfaces, including indwelling
medical devices such as hip joint
replacements
4) DARK FIELD MICROSCOPE

 In dark field microscope , the object

appears bright against a dark background.
 It is made possible by special dark field
condenser
 The dark field condenser has a central
opaque area that blocks light from entering
the object lens directly and has a peripheral
annular hollow area which allows the
light to pass through and focus on the
specimen obliquely.
DARK FIELD MICROSCOPE
 Uses of Dark Field Microscope
 It is useful for demonstration of
very thin bacteria not feasible
under
ordinary illumination
 Its is used for the demonstration of
motility of flagellated bacteria and
protozoa
 It is used to study mounted cells
and tissues.
5) BRIGHT FIELD MICROSCOPE

 Ordinary microscope is also

called bright field
microscope.
 It forms dark image against
light or bright background.
6) ELECTRON MICROSCOPE
 It was invented by German Physicist Ernst and Ruka in 1931.
 An electron microscope uses accelerated electrons as a
source of illumination
 Because the wavelength of electrons can be up to 100,000
time shorter than that of visible photons.
 The electron microscope has a much better resolving power
than a light microscope.
 Hence, it can reveal details of flagella, fimbriae and
intracellular structures of cell.
TYPES OF ELECTRON MICROSCOPE
Transmission Electron Microscope
Transmission Electron Microscopy (TEM) is a
technique of imaging the internal structure of solids
using a beam of high-energy electrons transmitted
through the solid.

Scanning Electron Microscope

A scanning electron microscope is a type of electron
microscope that produces images of a sample by
scanning the surface with a focused beam of
electrons.
7) PHASE CONTRAST MICROSCOPE

 Phase Contrast is a light microscopy technique used to enhance the

contrast of images of transparent and colorless specimens.
 It enables visualization of cells and cell components that would be
difficult to see using an ordinary light microscope.
PHASE CONTRAST MICROSCOPE

 It is used to produce high contrast

images of transparent specimens
such as living cells usually in
culture, microbes ,thin tissue slices.
8) FLUORESCENCE MICROSCOPE

 Refers to any microscope that uses fluorescence property to generate an

image
 When fluorescent dyes are exposed to UV rays, they become excited and
are said to fluoresce i.e. they convert this invisible , short wavelength rays
into light or longer wavelengths(visible light).
 The source of light may be mercury lamp which emits rays that pass
through an excitation filter.
 The exciting rays then get reflected by a dichromatic mirror in such a way
that they fall on the specimen which is priory stained with fluorescent dye.
FLUORESCENCE MICROSCOPE
REFERENCES

 https://www.ncbi.nlm.nih.gov/books/NBK555922/#:~:text=There%2
0are%20four%20major%20types,%2Dcoated%20plate%3B%20scre
ening%20antigen

 https://immunalysis.com/products/elisa/
 https://www.biologydiscussion.com/microscope/compound-microsco
pe-structure-and-working-principles/5822
 G. J. Tortora, B. R. Funke, C. L. Case, Microbiology: An
Introduction, Eleventh Edition ,Pearson, United States of America.