The document discusses antibody conjugation, a method of attaching antigens to larger molecules to enhance immune responses and generate antibodies. It details protocols for conjugation, common conjugates like KLH and BSA, and techniques to ensure proper orientation of antigens on carrier proteins. Additionally, it addresses the importance of purification steps for isolating specific antibodies that bind strictly to the target antigen.

1. Antibody Conjugation
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2. What is Antibody Conjugation ?
Conjugation is the method of adding an antigen to a larger
molecule that ensures that the antigen stimulates the immune
response that generates antibodies.

3. So, What is a conjugated antibody?
-Conjugated antibodies are high-performance monoclonal or polyclonal antibodies
coupled to a colormetric or fluorescent label for use as reporter molecules in
primary or secondary antibody immunoassays. Rockland conjugates are designed
to be used in a variety of applications, such as ELISA, FLISA, Western blot,
immunofluorescence, immunohistochemistry, flow cytometry, and others, while
working across multiple imaging platforms.
-A conjugated antibody is also known as a labeled, or tagged antibody.
It is one that has been bound to a substrate, such as an enzyme, toxin or
inorganic compound.

4. Why is Conjugation important?
B Lymphocytes develop antibodies to foreign antigens they encounter.
Nevertheless, the antigen must be significant enough for the immune system
to provide a humoral response – i.e. a reaction containing antibodies secreted
by B cells. Therefore, in order to generate an antibody for a particular antigen,
the researcher should ensure that the antigen is part of a significantly large
molecular complex to initiate a humoral response.

5. Protocol for Antibody Conjugation
1. Add 30 uL of 0.75 M sodium bicarbonate, pH 8.3, to 200 uL of primary antibody (label 5-8 tubes of
primary antibody to minimize waste of dye, as it loses stability when dissolved)
2. Dissolve the 1 mg vial of succinimidyl ester dye in 100 uL DMSO
3. Slowly add 12.5 – 20 uL of dye to each vial of primary antibody while vortexing the solution
4. Shake the tubes for 1 h at room temperature (we have used a bacterial shaker at 200 RPM)
5. Add 20 uL of freshly prepared 1.5 M hydroxylamine, pH 8.5 to each tube and shake for 1 h at
room temperature
6. Dialyze using 2 L of PBS in the dark at room temperature for 2 hour. We have then stored at 4
degrees Celsius without adding preservatives, but adding BSA and/or sodium azide is recommended
in the original protocol

6. Protocol for Antibody Conjugation
No protocol is given for antibody purification after conjugation (e.g., Protein x or Protein
z). In general, we do not purify our conjugates directed against cell-surface antigens,
since unreacted dye is removed during the washing steps. Our conjugates generally
have low background levels. However, you may find it useful to purify conjugates,
especially if they are to be used for intracellular (cytoplasmic) staining
The conjugations fall into four basic protocols: Type 1 (used for FITC, Cy5, and the initial
preparation of the Cy5 and Cy7 tandem dyes); Type 2 (used for Biotin and Cascade Blue);
Type 3 (used for conjugation of PE, APC, TR-BSA, and their derivatives); and Type 4 (used
for the initial preparation of non-immunoglobulin proteins like Annexin V). Buffers for all Type
1 reactions are identical, as for Type 2, etc.; some buffers are identical across the different
reaction protocols.

7. Popular Conjugates
The two most popular conjugates, also reffered as carrier proteins which antigens
bind to, are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
These two molecules are highly immunogenic, hence they are the best at
activating humoral response that creates antibodies directed against the antigen.
Another conjugate is ovalbumin (OVA), the main protein found in egg whites. OVA
is soluble in polar reagents, making it a strategic carrier for antigens that are also
polar.

8. Techniques for Exposing Specific Parts of the Antigen to the Immune System
The N-terminal and C-terminal of polypeptides contain distinct chemical
moeities that are ideal for various types of reactions that result in covalent
bonds. Moreover, the side-chains of the amino acid residues in the
polypeptide often have chemical moieties that can be used to create desired
covalent bonds with a carrier protein.This strategy allows antibody developers
to ensure that the antigen is oriented to the carrier protein in such a way that
the antibodies produced by the immune response recognize the desired
antigen epitope. Commonly used chemical cross-linkers used to affect the
orientation of antigens to carrier proteins are glutaraldehyde, carbodiimide,
benzidine and succinimide esters.

9. Steps to Isolate Antibodies That Bind Only to the Antigen
Since antigens attached to carrier proteins are engulfed and digested along
with carrier proteins, the antibodies produced by the immune response will
consist of antibodies that bind to different combinations of antigen carrier
fragments. This is problematic for an assay that involves a pure antibody
solution that only binds to the antigen. Extra purification steps need to be
taken in order to isolate the desired antibodies from those that recognize the carrier
protein only or a fragment that contains both antigen and carrier.

10. Thank You
From BosterBio (http://bosterbio.com/)