Download Antibody Production and Use in Immunodetection

Antibody Production
and Use in
Immunodetection
Introduction for ELISA and Western
Blotting Techniques
Papsin
Pepsin
FC region
Antibody(Immunoglobin-Ig
molecules
 Glycoproteins(MW-150kd)composed of
two identical heavy polypeptide chains
(MW- 50 kd) and two light chains (MW25kd each)
 The amino terminal of the chains show
variable amino-acid composition(V) and
the C-terminal regions are constant (C)
- Antigen
binds to the V region of the heavy and light chains
-Each Ig is bivalent and can bind two identical antigens
- Heavy and light chains are held together by non-covalent bonds
and covalent disulfide interactions
-The two heavy chains are held together by disulfide bonds at the
hinge region
-Treatment with proteolytic enzyme, pepsin generates a F(ab)2
fragment
-The enzyme, papsin, cleaves the Ig molecule at the hinge region
to generate two identical Fab fragments and a FC region.
IgG Classes
 The different Ig classes differ in their aminoacid sequences at the FC region
 1) IgG
 Has gamma heavy chains
 75% of the total Ig in human serum
 Secondary immune response in most antigens
 Can cross the placenta
 Most commonly used in biotech
2) – IgM
-Has Mu heavy chains
-Forms pentamers
-B-cell antigen receptor. Primary immune
response to most antigens
3) IgA
 Has alpha heavy chains
 Found in both monomeric and dimeric
forms
 Primary defense mechanism in
membrane secretions (eg. Tears and
saliva)
 Present in breast milk
Ig Classes
 Ig molecules also can be catagorized into
two subclasses depending on their type
of light chains:
 1) Kappa (k) light chain.
 2) Lambda heavy chain
Epitopes
Immunogen
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An antigen (non-self) that initiates an immune response
High MW > 6000d (6kd)
Chemical complexity
Proteins are strong immunogens
Carbohydrates with complex structure are
immunogenic
 Lipids are immunogenic
 Nucleic acids are poor immunogens
 Poor or non-imunogens can become immunogenic by
conjugation to a carrier protein (eg. BSA)
Inject antigen multiple times in
the presence of an immune
boosting compound called
adjuvant
Test for the presence of
antibodies and titer: The
Collect blood
samples
Centrifuge and collect the
serum
Recombinant Antibody
 An engineered recombinant DNAcoding
for an antibody molecule or the binding
portion of an antibody molecule is
inserted in bacteria,yeast or human cells
 The expressed antibody is pulled from
the cells
 No animal is used
Recombinant Antibody
 The technique can be used to make libraries of
antibody molecules with slightly different amino
acid sequences that can further tested to find
the one with the most affinity for an antigen
 The recombinant DNA can be engineered to
encode a human antibody molecule with the
binding portion of a mouse. This type of
antibody is called “humanized” or Chimeric
antibody. It eliminates the problem of using
animal antibodies on humans and triggering an
immune response.
Applications
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ELISA
Western Blotting
Immunochemistry
Immunoflouresence
Theraputic (Herceptin)