Basic Principles & Production of Antibodies

Separation of Bound and Unbound Drug.

PRESENTED BY

GOURAB DAS (170122885011)

MASTER’S OF PHARMACY 1ST SEMESTER

D E PA R T M E N T O F P H A R M A C E U T I C A L A N A LY S I S

G. PULLA REDDY COLLEGE OF PHARMACY

 CONTENTS…..

o INTRODUCTION OF IMMUNOASSAYS

o BASIC PRINCIPLES OF IMMUNOASSAY

o PRODUCTION OF ANTIBODIES

o SEPARATION OF BOUND DRUGS & UNBOUND DRUGS

 INTRODUCTION
• An immunoassay is an analytical technique used for quantification of an
ANALYTE based on Antigen-Antibody reaction.

• An antigen : antibody complex is also known as an immunocomplex.

• Here “immuno” refers an immune response that causes the body to generate
antibodies, and “assay” refers to a test. Thus, an immunoassay is a test that
utilizes immunocomplexing when antibodies and antigens are brought together.

• An immunoassay is an analytical method which use antibodies as reagent to

quantitate specific analyte.
 PRINCIPLES
• Immunoassay methods are based on a competitive binding reaction between a fixed
amount of labelled from an analyte and variable amount of unlabeled sample analyte
for a limited amount of binding site of a highly specific anti-analyte antibody.

• When the immuno-analytical reagents are mixed and incubated, the analyte is bound
to the antibody and forming an immune complex.
• The complex is separated from unbound reagent fraction by chemical and physical
separation technique.
• A standard curve ,which represents the measured signal which is a functional of the
concentration of the unlabelled analyte.
The advantage of immunoassay is that either the antigen or antibody can be labelled.

Types Of Immunoassay

 Radioimmunoassay (RIA)

 Enzyme-linked immunosorbent assays (ELISA)

 Fluoroimmunoasssay (FIA)

 Chemiluminescence immunoassay (CLIA)

 WHAT IS ANTIBODIES?
• An antibody is a protein used by the immune system to identify and neutralize the
foreign particle like bacteria and viruses. Each antibodies recognizes specific
antigen unique to its target.
• The high specificity of antibodies make then an a excellent tool for detecting and
quantifying a broad array of targets, from drug to serum protein to the
microorganism.
• With in vitro assay antibodies can be used to precipitated soluble antigens ,
opsonize and kill the bacteria with the assistance of complement and neutralize
drug toxin and virus.
• Antibody have specific
binding site to a specific
protein.

• Y Shaped Molecules
Which Are Generated By
Plasma Cells.

• Most Of The Material

Are Made Up Wi t h
Protein Sequence And
Some Carbohydrate
region.

• As they are response for

immunogen t h a t ’s why
we are called it as
“immuno”

• Albumin will migrate

and produced less
molecular weight
globular protein which
was further known as
globulin.
• “Fab” is responsible for antigen binding and “Fc” is the cellular & receptor binding site.
• Here 2 heavy chain and 2 light chain are present.(heavy chain – 440 amino acid long light chain – 220)
• The bonds are present as “Inter” & “Intra” chain. Where “Intra” refers to bond between themselves and ‘Inter” refers to
bond between different polypeptide chain.
• Two regions are present variable & continuous region. Variable region was 25% and continuous region was 75%.
• The FC region have major function like biological activity (binding, triggering receptor).
 Classification of antibodies

 MONOCLONAL ANTIBODIES

 POLYCLONAL ANTIBODIES

 ANTIBODY FRAGMENTS

 CHIMERIC ANTIBODIES

 HUMANIZED ANTIBODIES

 BISPECIFIC ANTIBODIES
MONOCLONAL ANTIBODY : Monoclonal antibodies are important reagents used in
biomedical research , in diagnosis diseases and treatment of such disease as infections and
cancer.
These antibodies are produced by cell illness or clones from the animal which has been
immunized with the subject that to be studied.

POLYCLONAL ANTIBODY : If an animal is immunized with a protein, a wide

array of B cell will be stimulated to produced anti-protein antibodies. They are a
combination of immunoglobulin molecules secreted against a specific antigen.
Polyclonal antibodies are a mixture of antibodies with different antigen binding sites
that may bind with different epitopes or antigen of the immunizing agents with
varying affinities.
Each identify a different epitopes.
ANTIBODY FRAGMENT: Earlier monoclonal antibodies are non human origin
which are immunogenic and exhibit human anti-immuno response. To overcome the
problem antibodies are cleaved into Fab and Fc fragments by papain digestion. The
Fab fragments are less immunogenic that intact antibodies.
CHIMERIC ANTIBODIES: It contains Fc region of human immunoglobulin (IgG)
and Fab regions are murine origin. These are slightly immunogenic.

HUMANIZED ANTIBODIES: These are produced by rRNA technology. Majority of

antibody framework is human in origin but the CDR(responsible for antigen binding) is
murine.

BISPECIFIC ANTIBODIES: Each of the arms, are specific for two different antigens.
For example: target1 and target 2.
What is monoclonal antibodies??
• Monoclonal antibodies are derived from a single clone of plasma cell, all having
the same antigen specificity.(which produced against single epitope of an antigen)
• *Epitope :- epitope is the immunologically active region of an immunogen that
binds to antigen specific membrane receptor on B-lymphocyte cell.
• Using produced in laboratory by using of hybridoma technology.
• Therapeutic monoclonal antibodies act through multiple mechanisms such as
blocking targeted molecule functions, inducing apoptosis in cell which express by
the target.
Steps in the production of Monoclonal antibodies
• The mouse is immunized by specific antigen injection against which monoclonal antibodies have to
be produced.

• After 72 hours immunization spleen is collected from the mouse (antibody producing B cell).

• The B cell are fused with immortal myeloma cells.

• The fused cells are incubated at HAT Medium.(hypoxanthine , aminopterin , thymidine)

• There are three possibilities - Unfused B cells

Unfused myeloma cells

Fused Hybridoma cells

Hybridoma :- fusion of two cells are generally known as hybridoma technique. Clone of B cell
stimulated against a single epitope of an antigen is fused with an immortal cell to produced a
hybridoma cell.
• FUSION:- the mouse splenic B –cell and
mutated myeloma cells are fused in
polyethylene glycol broth.

• Here B-cell can’t provide the antibodies

by their own.

• and the myeloma cell can’t grow

because of lack of hgprt enzyme and
thiamine kinases which was responsible
for production of purine synthesis.

• The spleen cell can grow in it but these

cells can’t survive.

• We are preparing the hybridoma by the

chemical known as HAT. In that solution
the myeloma cells can’t survive .
SELECTION OF INDIVIDUAL CELLS

• The original antigen used to has multiple epitopes many B cells are fused with
myeloma all to proceeded mixture of hybridoma cells .

so the cells having specificity for one epitopes.

• Medium contain hybridoma cell then dilute into multi well plant to search extent
that each well contains only one cells.

• Hybridoma tells to produced desired monoclonal antibodies are selected by RIA

and ELESA using antigen fragment.
What is polyclonal antibodies??
• A polyclonal antibody is a collection of many immunoglobulins each generated from a different
B-cell clone. These antibodies targets different epitopes ,or binding sites on a single antigen.

• Polyclonal antibodies are heterogeneous mixture which are generally produced by different B-cell
clones in the body. They can recognize and bind to many different epitopes of a single antigen.

• Each antibody recognizes different epitopes.

• Polyclonal antibodies refers to a mixture of immunoglobulin molecules which are secreted against
a particular antigen.

• This means polyclonal antibodies are heterogeneous mixture of antibodies.

Steps in the production of Polyclonal antibodies

• All antibody generation, polyclonal or monoclonal antibodies begins with eliciting

an immune response which was termed as immunization.

• The intended targets are injected into the host, recognized by the immune system
as foreign ,and targeted by antibodies for immune blockage or clearance.

• Failure to mount an immune response would required the use of adjuvants,

immunogenic substance such as Freund’s adjuvant. Immunization comprises the
majority of what is required to make polyclonal antibodies.
General steps for production of polyclonal antibodies

• Polyclonal antibodies are often injecting a lab animal most commonly used as
rabbits, goats and mouse with a specific antigen, to devolve the antibody.

• We inject the antibodies inside our organism.

• After a weeks (mostly six weeks) the immune system of animal starts producing
the high level of antibodies specific for the antigens.

• The plasma cells, present inside the body of that animal starts producing
antibodies.
• Then we extract these antibodies from the body of that animal and purify all the
antibodies.

• Normally, in this case antibodies which are produced are IgG variations

• Typical methods of exposure include injection into the skin, peritoneum,

subcutaneous layer or muscle.

• DNA immunization also an emerging technology for toxic or difficult to produced

antigens. Any subsequent exposure to antigen is termed a “boost” immunization.
Initial exposure for primary
response

Antigen processing by
antigen processing cell

stimulation of B-cell

Proliferation of B-cell and

antibody secretion

plasma cell development

Purification process
This step basically multi-step
process which was include:-
• Sample preparation
• Capture
• Initial purification
• Secondary purification
• Polishing and formation
 ANTIBODY – ANTIGEN RACTIONS

• Each antibody has the ability to bind to a different foreign agent, or antigen (Ag)

• The ability of an antibody to recognize and bind a given antigen depends on the structure of its

binding site

• Determined by the amino acid sequence of the antibody near the N-terminal ends of the heavy and

light chains

• The general reaction between a single binding site on the antibody (Ab) and antigen (Ag) can be

written as follows:

Ab + Ag Ab Ag

• The binding is very selective and only occurs between Ab and Ag, or between Ab and molecules

similar to Ag in their three-dimensional structure.

 ANTIBODY USAGE

• The selectivity of Ab-Ag interaction makes antibodies useful as analytical

reagents for the determination of specific components in mixtures. Antibodies are
useful as analytical reagents since they can be produced to a wide variety of
substances: For large analytes (> 5,000 MW), antibodies can be produced by
directly injecting the compound into an animal. For small analytes (< 5,000 MW),
antibodies can also be produced, but require that the compound first be coupled to
a larger molecule, such as a protein, prior to injections.
SEPARATION OF BOUND AND UNBOUND DRUGS

• LIQUID PHASE METHODS

1. Adsorption:
These methods are based on differential adsorption. Following sedimentation of the adsorbent,
the bound drug is decanted and free are counted for radio isotopic or enzymatic activity or
fluorescence Concerns about misclassification errors resulting from dependence on time,
temperature, buffer, ionic strength conditions has caused this modality to become less popular.
2. Non specific precipitation:
Addition of salt or solvent that changes the solubility properties and causes them to precipitate
from solution. Caution must be exercised when using these methods because of non specific
nature.
3. Specific precipitation:
Addition of second antibody causes lattice formation with primary antibody and
immune complexes precipitate. PEG may be added to the double antibody mixture to
increase the rate of reaction shorten incubation times required for successful separation
of free and bound.Less widely employed methods of separation include molecular size
chromatography such as microfiltration, affinity column chromatography and
electrophoresis.

• SOLID PHASE METHODS

1.Mobile solid phase reagents:

The carbohydrate particles are readily with cyanogen bromide to covalently couple
protein. To counter centrifugation drawback, ferromagnetic particles have been
developed.
2.Stationary solid phase reagents:
These have been developed to eliminate centrifugation. Micro-techniques of coating
antibody on capillary tubes, glass rods, chromatography tubes are widely employed.
Coating of plastic plates, tubes, flat surfaces with antigens or antibodies is also
widely used. Criticism of coated plastic solid phases concerns the adsorption of
molecules onto a surface in a configuration which can mask immunoreactive
determinants and reduce immunoreactivity. Plates and tubes have limited binding
capacity for protein.
 REFERENCES….

• Pharmaceutical biotechnology by Dr. S.P.VYAS, CBS publishers page no.485.

• Textbook of microbiology by Dr. C S Bhaskaran, universities press page no.133.
• Pharmaceutical biotechnology by S.S. Purohit page no. 103.
• Pharmaceutical biotechnology by Ashutosh kar page no. 238.
• Textbook of pharmaceutical microbiology by Prescott page no.655.