BCH 408: IMMUNOCHEMISTRY (2 Credit Units)

BY: PROFESSOR FAOZIYAT ADENIKE SULAIMAN

COURSE CONTENTS:
-Production of specific (monoclonal and poly clonal antibodies)

-Enzyme-linked immunosorbent assay (ELISA); Radioimmunoassay; Haemagglutination

and blood group serology

-Application of immunochemistry in clinical practice and biological research

VISUAL TEACHING AIDS

Monoclonal Antibodies
https://www.bing.com/videos/riverview/relatedvideo?&q=production+of+polyclonal+antibodies&&m
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https://www.youtube.com/watch?v=vhT7SQaFS6E

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Polyclonal Antibodies
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Monoclonal versus Polyclonal

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Monoclonal antibodies
Monoclonal antibodies are artificial antibodies produced from a single clone of cells using a
technique called hybridoma technology. Let me explain how this process works:

1. Immunization: An antigen (a foreign protein) is injected into an animal, typically a

mouse. This stimulates the production of lymphocytes (immune cells) that generate
antibodies specific to the antigen.
2. Cell Fusion: Spleen cells containing the antibody-producing lymphocytes are removed
from the immunized animal. These spleen cells are then fused with immortalized
myeloma cells (cancerous white blood cells) to create hybrid cells called hybridomas.
3. Selection and Expansion: Hybridomas are cultured under specific conditions where
unfused normal cells and tumor cells cannot survive. Each hybridoma produces a single
type of antibody derived from a specific B cell. Researchers screen these hybridomas to
find the one that secretes antibodies with the desired specificity.
4. Monoclonal Antibody Production: The selected hybridoma is expanded, and the
antibodies it produces are harvested. These antibodies are monoclonal, meaning they
recognize a single epitope (binding site) on the antigen used for immunization.
Monoclonal antibodies have various applications, including targeted therapy for cancer,
autoimmune diseases, and diagnostics. They are powerful tools in research and medicine due to
their specificity and consistency.

The production of monoclonal antibodies is an in vitro process by the use of tissue culture
techniques. Producing monoclonal antibiotics (mAbs) is initially done by identifying a specific
antigen.

Types of Monoclonal Antibiotics: Being synthetically manufactured to act like human antibodies in the
immune system, monoclonal antibodies are prepared in 4 different ways and they are named after what they
are made of.

Functions and Applications of monoclonal antibodies

 1. Monoclonal antibodies are used in the treatment of several diseases and disorders.
 2. Some diseases and disorders treated using mAbs include: Cancers, Rheumatoid.
 3. Monoclonal antibodies are widely used in therapies, laboratory investigations, …etc

What Are Monoclonal Antibodies?

 1. The body naturally produces antibodies, which are elements of the immune system produced by B-
lymphocytes, that bind to …
 2. They naturally circulate in the body searching for foreign bodies (antigens) and once they attach to
the antigen , a complex is formed.

Polyclonal antibodies
Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body
(whereas monoclonal antibodies come from a single cell lineage). They are a collection
of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope.

Production
The general procedure to produce polyclonal antibodies is as follows:

1. Antigen preparation
2. Adjuvant selection and preparation
3. Animal selection
4. Injection process
5. Blood serum extraction
An antigen/adjuvant conjugate is injected into an animal of choice to initiate an amplified immune response.
After a series of injections over a specific length of time, the animal is expected to have created antibodies
against the conjugate. Blood is then extracted from the animal and then purified to obtain the antibody of
interest.
Inoculation is performed on a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often
preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This
induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is
purified from the mammal's serum.
By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines
governing animal use and procedures relating to these methodologies are generally oriented around humane
considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while
having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and
sites of administration, injection volumes per site and number of sites per animal. Institutional policies
generally include allowable volumes of blood per collection and safety precautions including appropriate
restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.
The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for
use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to
antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen
into draining lymph nodes.
Many adjuvants also contain or act directly as:

1. surfactants which promote concentration of protein antigens molecules over a large surface
area, and
2. immunostimulatory molecules or properties. Adjuvants are generally used with soluble
protein antigens to increase antibody titers and induce a prolonged response with
accompanying memory.
Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple
B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein
antigens are generally poorly or enhanced by adjuvants and there is no system memory.
Antibodies are currently also being produced from isolation of human B-lymphocytes to produce specific
recombinant monoclonal antibody mixtures. The biotechnology company, Symphogen, develops this type of
antibodies for therapeutic applications. They are the first research company to reach phase two trials with the
monoclonal antibody mixtures that mimic the diversity of the polyclonal antibody drugs. This production
prevents viral and prion transmission and this is the simple process.
Animal selection
Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters,
horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this
purpose. Animal selection should be based upon:

1. the amount of antibody needed,

2. the relationship between the donor of the antigen and the recipient antibody producer
(generally the more distant the phylogenetic relationship, the greater the potential for high
titer antibody response) and
3. the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the
antibodies to be made. Immunization and phlebotomies are stress associated and, at least
when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of
such animals can dramatically reduce morbidity and mortality due to pathogenic organisms,
especially Pasteurella multocida in rabbits.
Goats or horses are generally used when large quantities of antisera are required. Many investigators favor
chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG)
into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure.
One week's eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one
weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in
immunoassays. Chicken IgY does not fix mammalian complement component C1 and it does not perform as a
precipitating antibody using standard solutions.
Although mice are used most frequently for monoclonal antibody production, their small size usually prevents
their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can
be collected from ascites fluid using any one of a number of ascites producing methodologies.
When using rabbits, young adult animals (2.5–3.0 kg or 5.5–6.5 lbs) should be used for primary immunization
because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new
antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported
to mount a more vigorous immune response than males. At least two animals per antigen should be used when
using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to
antigens of individual animals.
Antigen preparation
The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality
and quantity of antibody produced. Small polypeptides (<10 ku) and non-protein antigens generally need to be
conjugated or crosslinked to larger, immunogenic, carrier proteins to increase immunogenicity and provide T
cell epitopes. Generally, the larger the immunogenic protein the better. Larger proteins, even in smaller
amounts, usually result in better engagement of antigen presenting antigen processing cells for a satisfactory
immune response. Injection of soluble, non-aggregated proteins has a higher probability of inducing tolerance
rather than a satisfactory antibody response.
Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-
lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces
or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody
production. Recently, liposomes have also been successfully used for delivery of small peptides and this
technique is an alternative to delivery with oily emulsion adjuvants.
Antigen quantity
Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant
selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer
antibodies. Antigen dosage is generally species, rather than body weight, associated. The so-called “window”
of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression
or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual
protein antigen levels for immunizing specific species have been reported in the following ranges:

1. rabbit, 50–1000 μg;

2. mouse, 10–50 μg;
3. guinea pig, 50–500 μg; and
4. goat, 250–5000 μg.
Optimal “priming” doses are reported to be at the low end of each range.
The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and
as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are
usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and
chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.
Peptide antibodies
When a peptide is being used to generate the antibody, it is extremely important to design the antigens
properly. There are several resources that can aid in the design as well as companies that offer this service.
Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user
knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that
will enable you to score individual peptide sequences based upon a relation epitope mapping database of
previous immunogens used to generate antibodies. Finally, as a general rule peptides should follow some basic
criteria.
When examining peptides for synthesis and immunization, it is recommended that certain residues and
sequences be avoided due to potential synthesis problems. This includes some of the more common
characteristics:

 Extremely long repeats of the same amino acid (e.g. RRRR)

 Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets
 Ending or starting a sequence with a proline (P)
 Glutamine (Q) or Asparagine (N) at the n-terminus
 Peptides over weighted with hydrophobic residues (e.g. V, A, L, I, etc....)
Reactivity
Investigators should also consider the status of nativity of protein antigens when used as immunogens and
reaction with antibodies produced. Antibodies to native proteins react best with native proteins and
antibodies to denatured proteins react best with denatured proteins. If elicited antibodies are to be used on
membrane blots (proteins subjected to denaturing conditions) then antibodies should be made against
denatured proteins. On the other hand, if antibodies are to be used to react with a native protein or block a
protein active site, then antibodies should be made against the native protein. Adjuvants can often alter the
nativity of the protein. Generally, absorbed protein antigens in a preformed oil-in-
water emulsion adjuvant, retain greater native protein structure than those in water-in-oil emulsions.
Asepticity
Antigens should always be prepared using techniques that ensure that they are free of microbial contamination.
Most protein antigen preparations can be sterilized by passage through a 0.22 μm filter. Septic abscesses often
occur at inoculation sites of animals when contaminated preparations are used. This can result in failure of
immunization against the targeted antigen.
Adjuvants
There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types varies
depending upon whether they are to be used for research and antibody production or in vaccine development.
Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those
for antiserum production need to rapidly induce high titer, high avidity antibodies. No single adjuvant is ideal
for all purposes and they all have advantages and disadvantages. Adjuvant use generally is accompanied by
undesirable side effects of varying severity and duration. Research on new adjuvants focuses on substances
which have minimal toxicity while retaining maximum immuno-stimulation. Investigators should always be
aware of potential pain and distress associated with adjuvant use in laboratory animals.
The most frequently used adjuvants for antibody production are Freund's, Alum, the Ribi Adjuvant System,
Specol and Titermax.
Freund’s adjuvants
There are two basic types of Freund's adjuvants: Freund's Complete Adjuvant (FCA) and Freund's Incomplete
Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is
formulated with mineral oil, the surfactant mannide monoleate and heat killed Mycobacterium
tuberculosis, Mycobacterium butyricum or their extracts (for aggregation of macrophages at the inoculation
site). This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of
antibody against epitopes of denatured proteins. Although FCA has historically been the most widely used
adjuvant, it is one of the more toxic agents due to non-metabolizable mineral oil and it induces granulomatous
reactions. Its use is limited to laboratory animals and it should be used only with weak antigens. It should not
be used more than once in a single animal since multiple FCA inoculations can cause severe systemic reactions
and decreased immune responses. Freund's Incomplete Adjuvant has the same formulation as FCA but does
not contain mycobacterium or its components. FIA usually is limited to booster doses of antigen since it
normally much less effective than FCA for primary antibody induction. Freund's adjuvants are normally mixed
with equal parts of antigen preparations to form stable emulsions.
Ribi Adjuvant System
Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil
(squalene) which are then emulsified with saline containing the surfactant Polysorbate 80. This system also
contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial
monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available.
These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances
antigen uptake, processing and presentation. This adjuvant system is much less toxic and less potent than
FCA but generally induces satisfactory amounts of high avidity antibodies against protein antigens.
Titermax
Titermax represents a newer generation of adjuvants that are less toxic and contain no biologically derived
materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers
polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other
surfactant materials and have potent adjuvant properties which favor chemotaxis, complement activation and
antibody production. Titermax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and
metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for
incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms
hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major
histocompatibility molecules on macrophages. Titermax presents antigen in a highly concentrated form to
the immune system, which often results in antibody titers comparable to or higher than FCA.
Specol: Specol is a water in oil adjuvant, made of purified mineral oil. It has been reported to induce immune
response comparable to Freund's adjuvant in rabbit and other research animal while producing fewer
histological lesions.
Pharmaceutical uses
-Digoxin Immune Fab is the antigen binding fragment of polyclonal antibodies raised to Digitalis derivative
as a hapten bound to a protein and is used for the reversal of life-threatening digoxin or digitoxin toxicity.
-Rho(D) immune globulin is made from pooled human plasma provided by Rh-negative donors with
antibodies to the D antigen. It is used to provide passive immune binding of antigen, preventing a maternal
active immune response, which could potentially result in hemolytic disease of the newborn.
-Rozrolimu pab is the anti-RhD recombinant human polyclonal antibody composed of 25
unique IgG1 antibodies and is used for the treatment of immune thrombocytopenia purpura and prevention of
isoimmunization in Rh-negative pregnant women.
-REGN-COV2 (Regeneron CoV2) – potential treatment for people with COVID-19 and to prevent SARS-
CoV-2 coronavirus infection.
Advantages of polyclonal antibiotics
The use of polyclonal antibodies (PAbs) over monoclonal antibodies has its advantages.1) The technical skills
needed to produce polyclonal antibodies is not as demanding. 2) They're inexpensive to make and can be
generated fairly quickly, taking up to several months to produce. 3) PAbs are heterogeneous, which allows
them to bind to a wide range of antigen epitopes.4) Because PAbs are produced from a large number of B cell
clones, they're more likely to successfully bind to a specific antigen. 5) PAbs remain stable in different
environments, such as a change in pH or salt concentration, which allows them to be more applicable in certain
procedures. 6) Additionally, depending on the amount needed, PAbs can be made in large quantities in relation
to the size of the animal used.

-Enzyme-linked immunosorbent assay (ELISA); Radioimmunoassay; Haemagglutination

and blood group serology
-Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a test that detects and measures antibodies,
antigens, proteins and glycoproteins in biological samples. ELISA uses antibodies to bind to to
a target antigen and an enxyme to produce a colour change that indicates the presence and
quantity of the antigen. ELISA is used for diagnosis of various diseases, such as HIV
infection, pregnancy, and cytokine levels.

This is how the enzyme-linked immunosorbent assay (ELISA) works:

1. Coating Phase:
o A microplate (usually 96 wells) is coated with a capture antibody specific to the
target antigen. The antibody adheres to the surface of the well.
o The sample (containing the antigen) is added to the wells and incubated. If the
antigen is present, it binds to the immobilized antibody.
2. Washing Phase:
o Unbound components are washed away to remove any non-specific interactions.
3. Detection Phase:
o A detection antibody (labeled with an enzyme) is added. This antibody recognizes
a different epitope on the antigen.
o If the antigen is present, the detection antibody binds to it.
4. Substrate Addition:
o A substrate containing the enzyme’s substrate is added.
o The enzyme catalyzes a reaction, resulting in a color change (usually a
chromogenic substrate turns from colorless to colored).
5. Measurement:
o The intensity of the color change is proportional to the amount of antigen present.
o The absorbance is measured using a spectrophotometer.
6. Quantification:
o A standard curve (using known concentrations of the antigen) helps quantify the
unknown sample’s antigen concentration.

ELISA variants include direct, indirect, sandwich, and competitive ELISAs, each with specific
applications. Researchers and clinicians use ELISA for diagnosing diseases, detecting antibodies,
and measuring protein levels.

There are four main types of ELISA, each with unique advantages and applications:
1. Direct ELISA:
o The antigen is immobilized directly onto the multi-well plate surface.
o Detection occurs using an antibody specific to the antigen, which is directly
conjugated to a detection molecule (e.g., HRP).
o Advantages: Simplicity, quick results, and suitability for detecting antigens.
 o Disadvantages: Limited antigen information and temporary readouts
2. Indirect ELISA:
o A two-step process: Primary antibody binds to the antigen, followed by a labeled
secondary antibody against the primary antibody.
o Advantages: High sensitivity, flexibility, and ability to detect antibodies.
o Disadvantages: Requires more steps and time
3. Sandwich ELISA:
o The antigen is captured between two antibodies (one immobilized and one
labelled).
o Used for quantifying antigens (e.g., cytokines) in samples.
o Advantages: High specificity, quantitative results, and suitability for complex
samples.
o Disadvantages: Requires specific antibody pairs
4. Competitive ELISA:
o Involves competition between labeled and unlabeled antigens for binding to a
limited number of antibodies.
o Used for small molecules (e.g., hormones, drugs).
o Advantages: Quantitative, adaptable, and suitable for low-affinity interactions.
o Disadvantages: Complex setup and interpretation

The choice of ELISA type depends on the specific assay requirements and the target antigen.

Limitations: The enzyme-linked immunosorbent assay (ELISA) has several limitations:

1. Complex Assay Workflows:
o ELISA protocols involve multiple time-intensive wash steps, making them
laborious and less suitable for high-throughput screening.
o Alternative immunoassays (e.g., flow cytometry, Meso Scale Discovery, Luminex
Bead-Based assays) offer streamlined protocols without extensive wash steps
2. Single End-Point Analysis:
o ELISA allows testing of only one analyte at a time. To test multiple analytes,
separate assays are required.
o In contrast, other methods (e.g. Electrochemiluminescence, AlphaLISA
multiplexed flow cytometry) enable simultaneous testing of multiple analytes in a
single assay.
3. Limited Dynamic Range:
o ELISA has a restricted quantification range, leading to saturation or detection
limitations.
o This limitation can result in inaccurate measurements.
4. False Positives and Negatives:
o ELISA can yield false positives or false negatives due to assay conditions or
sample characteristics.
o Ensuring proper inhibition of immunogenic antigens is crucial to avoid false
results
o
5. Antibody Instability:
o ELISA antibodies are proteins and require refrigerated storage and transport.
 o Preparing antibodies for ELISA can be labor-intensive and costly

Despite these limitations, ELISA remains a valuable tool in Biomedical research, but researchers
often explore alternative immunoassays to address specific challenges.

Advantages: The enzyme-linked immunosorbent assay (ELISA) offers several advantages:

1. High Sensitivity and Specificity: ELISA is sensitive and specific in detecting antibodies
and antigens, making it reliable for diagnosing infections and other conditions.
2. Simple Procedure: ELISA is straightforward to perform, and it doesn’t require
complicated pre-treatment steps.
3. Efficiency: Simultaneous analysis can be done without extensive sample purification,
making ELISA efficient.
4. Cost-Effective: ELISA assays are cost-effective and widely used in research and clinical
diagnosis.
5. Rapid Results: ELISA provides quick results, which is valuable for timely diagnosis.
ELISA is considered the gold standard for immunoassays, and its accuracy contributes to its
widespread use in various medical tests.

The differences between direct and indirect ELISA:

1. Direct ELISA:
o Single Antibody: In direct ELISA, only one antibody is used.
o Detection Method: The primary antibody is directly conjugated to the detection
enzyme (e.g., HRP or fluorophore).
o Advantages:
 Saves time due to the single-step process.
 Cost-effective (requires only one antibody).
o Use Case: Detecting antigens with a specific antibody.

2. Indirect ELISA:
o Two-Step Process: Indirect ELISA involves two antibodies.
o Primary Antibody: The primary antibody (specific to the antigen) binds to the
target.
o Secondary Antibody: A labeled secondary antibody (against the host species of
the primary antibody) binds to the primary antibody for detection.
o Advantages:
 Amplification step enhances sensitivity.
  Versatile and widely used.
o Use Case: Detecting antibodies or quantifying antigens.

Both methods have their merits, and the choice depends on the specific assay requirements.

3). A sandwich ELISA is an antibody-based technique used to quantify specific proteins,

hormones, or other analytes in a sample. Here’s how it works:

1. Principle:
o The target protein (antigen) is “sandwiched” between two antibodies: a capture
antibody and a detection antibody.
o The capture antibody binds to one epitope on the antigen, while the detection
antibody recognizes a different epitope.
2. Procedure:
o Coating: Coat a microtiter plate with the capture antibody at 1–10 μg/mL
concentration in carbonate/bicarbonate buffer (pH 9.6).
o Blocking: Block remaining protein-binding sites with a blocking buffer (5% non-
fat dry milk/PBS).
o Sample Addition: Add diluted samples containing the antigen.
o Detection Antibody: Add the detection antibody (labeled with an enzyme).
o Substrate Addition: Enzyme catalyzes a reaction, producing a color change.
o Quantification: Measure the color intensity (proportional to antigen
concentration).
o
3. Advantages:
o Removes sample purification step.
o Enhances sensitivity (2–5 times more sensitive than direct or indirect ELISA).
o Useful for complex samples and quantifying small differences in antigen levels.

Using well-matched antibody pairs is crucial for accurate results in sandwich ELISA.

NEXT LECTURE

-Radioimmunoassay
-Haemagglutination and blood group serology

-Applications of immunochemistry in clinical practice and biological research