| 1 |
How long does it take to make antibodies by GENOVAC's
technology? |
| |
For standard proteins it takes 8-12 weeks to generate
polyclonal antibodies. For monoclonal antibodies we need
4-6 months; for multiple-membrane spanning proteins it
may take up to 9 months. |
| 2 |
What guarantee is provided (internal controls)? |
| |
All services are provided by us using recognised scientific
procedures. However, owing to the nature of the services,
which are performed using living organisms with all their
inherent limitations, we cannot give any guarantee of
success. If during the fulfilment of an individual experimental
step it appears that the generation of a specific antibody
using the cDNA concerned becomes highly improbable, GENOVAC
will consult with the customer on further progress. However,
services already fulfilled by GENOVAC are not affected
by the possible omission of ongoing services. To minimise
the risk, each project is divided into several single
steps:
1. Feasibility study based on the cDNA sequence and antigen
information
2. Expression control of the immunisation construct by
cell surface expression
3. Immunisation and testing of specificity using cell
surface fluorescence
4. Fusion and Screening of hybridoma supernatants for
the presence of specific antibodies using CELISA
5. Subcloning testing of specific antibodies using FACScan
6. Delivery of the positive hybridomas and corresponding
supernatant |
| 3 |
What are your ideal targets? |
| |
Secreted or membrane-bound targets, because they are
naturally routed out of the cell. In order to stimulate
an immune response, all protein constructs are brought
out of the cell so that they can be recognised by the
immune system. For example, intracellular proteins are
not normally routed out of the cell, however, we also
have a high success rate against this class of proteins,
despite re-routing. Our overall success rate is 84%. |
| 4 |
Do I need to provide the cDNA? |
| |
In general, we receive the cDNA from our customers,
but we are also able to synthesise DNA. Usually we only
need 5-10 microgram's of the cDNA. |
| 5 |
What is the min/max size of the DNA? |
| |
The minimum size is usually determined by conformational
domain size. We prefer to choose larger constructs which
will allow conservation of the native conformation. We
can generate and immunise with peptide-encoding sequences,
but would recommend synthetic peptides as a cheaper approach.
We normally work with constructs ranging from approx.
75bp - 2500bp. Larger constructs are also possible. Our
largest are approximately 4000 bp. |
| 6 |
What is your success rate? |
| |
Our overall success rate is currently 84%. |
| 7 |
Do you have scientific publications? |
| |
Many of our clients are companies and usually they do
not publish their results. However, a list of available
scientific publications can be offered upon request. |
| 8 |
Do you have reference customers? |
| |
A list of reference customers can be provided upon request. |
| 9 |
Can you use other animals? |
| |
We immunise mice, rats or rabbits. |
| 10 |
Do you have glycosylation with intracellular targets? |
| |
Yes, if putative N-glycosylation sites exist in the
construct. These are avoided when possible in developing
the constructs. |
| 11 |
Can you generate antibodies to differentiate phosphorylated
targets? |
| |
Not with genetic immunization, but with phospho-peptides. |
| 12 |
Can you make antibodies against conformational
and linear epitopes? |
| |
Yes, but there is a preference towards conformational
epitopes. This is very dependent on the antigen in question
and cannot be predicted in advance. |
| 13 |
Can you generate antibodies against artificial epitopes? |
| |
If a DNA sequence is known, yes. Otherwise, no. |
| 14 |
Do the antibodies work in Western Blot? |
| |
Our antibodies are generated to recognise native proteins.
We cannot guarantee that they work in Western Blot, where
denatured proteins are presented. Sometimes they work,
sometimes they do not work, indicating a strong preference
for antibody generation against conformational epitopes. |
| 15 |
Do you offer affinity purification of polyclonal antisera? |
| |
We cannot purify polyclonal antisera using our transiently
transfected cells, i.e. to affinity purify antibodies
specific for the target. However, we can purify immunoglobulin
fractions, if required. |
| 16 |
Can you determine the isotype of the antibodies? |
| |
Yes. This is part of the final quality control before
delivery of the hybridoma cells. |
| 17 |
Do you offer monoclonal antibody purification? |
| |
Yes. We can purify IgG, IgA or IgM. |
| 18 |
What is the usual titer you receive? |
| |
1:10,000 - 1: 50,000, as determined by dilution ELISA.
We can go higher by extending the immunisation protocol,
if required. |
| 19 |
What is the usual affinity (KD) you get? |
| |
Thus far, we have only been able to test the affinity
in cases where the customer has provided a recombinant
antigen to test affinity. In these cases, KD values were
in the subnanomolar range. |
| 20 |
Can you break tolerance? |
| |
Currently no. However, we have developed proprietary vectors which helps us to evoke an immune response against highly homologous proteins |
