|
CUSTOMIZED
DEVELOPMENT
 |
Lateral
Flow Test Development: |
| ............................................................................................................. |
| |
 |
Outline
of the Lateral Flow Test Development Process |
|
Strategy
Planning |
| |
|
Positive
Antibody Selection |
| |
|
Characterization
of Best Conditions for Colloidal Gold Conjugation and NC Membrane
Coating for Each Antibody |
| |
|
Best
Matched Pair Antibody Selection |
| |
|
Development
of Rapid Test |
| |
|
Finalization
of Optimized Conditions |
| |
|
Finalize
Test |
| |
|
|
Monoclonal
Antibodies |
| ............................................................................................................. |
| |
|
Immunization |
| |
|
Cell
Fusion and Screening |
| |
|
Cloning |
| |
|
Antibody
Production |
| |
|
Antibody
Purification |
| |
.............................................................................................................................................................
Lateral Flow Test Development:
In
addition to developing and manufacturing our own rapid tests, we
also develop new and innovative rapid tests for our clients. The
development of a rapid test is a difficult and time-consuming process
even for very experienced researchers, and thus leaving the work
to Artron BioResearch, a leader in the development of rapid tests,
may be beneficial for your company.
In
developing a test for an antigen, one of the most important requirements,
and the most difficult to obtain, is a matching pair of monoclonal
antibodies (one for coating and the other for conjugating to colloidal
gold) that will give the test the required specificity, sensitivity,
and cross reactivity. The exact environment/conditions for this
antibody pairing also has to be determined, as an antibody pair
that matches requirements in one environment may not be active at
all in another. The client may provide the monoclonal antibodies
for this development (usually requiring about 10 clones), or may
contract us to develop the monoclonal antibodies by providing us
the antigen (see Custom Processes: Monoclonal Antibodies for details).
The exact conditions for coating, conjugating, washing, blocking,
the type of materials/buffers used in the sample pad, and the assembly
of the rapid test can also vary greatly between different tests.
Thus, due to the complexity of the process, pricing of the rapid
test development can vary greatly. An outline of the steps in the
development process is described below, and the client will be updated
on every step and no step will proceed without client approval.
Please do not hesitate to contact us with questions regarding development,
or to set up a meeting with our senior researchers.
.............................................................................................................................................................
Outline
of the Lateral Flow Test Development Process:
1.
Strategy Planning:
Senior scientists will meet with the client to discuss the possibilities
and limitations regarding the type of test the client wishes to
develop. A detailed proposal of development plans will then be provided
for the clientĄ¯s approval, and once approved, scientists and technicians
will be assigned to the project. A confidentiality agreement will
be signed at this point.
2.
Positive Antibody Selection:
Approximately 150 different clones that test positive (by ELISA)
for antibody activity will be required for development. The client
can choose to provide us with the selected antibodies (purified),
or to have us develop and select the hybridoma clones. Reactivity,
specificity, and cross-reactivity will be characterized for each
clone.
Note: because some antibodies could end up being discarded from
the development process due to undesired cross-reactivity or a low
level of reactivity and/or sensitivity, we may need more than 150
clones.
3.
Characterization of Best Conditions for Colloidal
Gold Conjugation and NC Membrane Coating for Each Antibody:
The best procedures and conditions for conjugating and coating antibodies
differ for each antibody and will need to be accurately characterized
before the extensive pairing process begins.
4.
Best Matched Pair Antibody Selection:
Extensive comparison studies will take place by pairing all antibodies
in different combinations (as the conjugate, or as the coating antibody,
with about 5000 pairs in total for trial). 2 pairs that meet requirements
of specificity, sensitivity, and cross-reactivity will be selected.
Note: it is entirely possible that no matching pairs will be found,
in which case, cell fusion will need to be repeated, 150 new positive
clones selected, and the selection and characterization process
repeated. Although this scenario only occurs in the most unfortunate
cases, the client should be financially prepared for its occurrence.
5.
Development of Rapid Test:
From the 2 pairs of antibodies, tests will be made with different
blocking and washing methods, buffers, support membranes, and sample
pads to optimize the reaction conditions on the test strip.
6. Finalization of Optimized Conditions:
Advanced technology for the production of lateral flow tests is
key to quality. Since Artron BioResearch already possesses advanced
technology for manufacturing test strips, finalizing the optimal
conditions for your customized test will hardly be a challenge.
Examples of parts of production process that will be finalized /
optimized are:
a.
conditions for conjugating MAb to colloidal gold
b. storage conditions for the conjugates
c. coating conditions for MAb and goat anti-mouse IgG
d. solutions and methods of blocking and washing NC membranes
e. type of supporting membrane (M2, M3, or M5)
At the end of production optimization, 2 prototype tests will be
made from the 2 pairs of antibodies selected to undergo detailed
field trials (client-conducted if desired).
7.
Finalize Test:
The better of the two prototypes will be selected based on sensitivity,
specificity, and cross-reactivity requirements. The client will
be given final documentation package of the entire process. All
client-supplied antigens, antibodies, materials, and intellectual
properties will be returned to the client. All research documents
compiled by Artron BioResearch for the project and all tests made
are property of the client.
.............................................................................................................................................................
Monoclonal
Antibodies:
(mouse hybridoma technology)
Artron
BioResearch Inc. has been using hybridoma technology to produce
monoclonal antibodies since its incorporation. Should the client
desire custom monoclonal antibodies to be developed against a provided
antigen, our experienced and talented researchers can meet with
the client to discuss possibilities. Each step of the process will
be thoroughly discussed with the client to ensure satisfaction.
Pricing can vary according to the nature of the antigen and the
complexity of the process. The general steps and our methods are
discussed below:
1.
Immunization
With the client-specified antigen (of which our company can assist
with purification as higher levels of purity usually improves the
overall success of generating useful antibodies), mice will be immunized
with either a client-specified method or our own recommended method.
A senior scientist, upon reception of the client request and antigen
nature, will determine the immunization process details and report
to the client for approval (conjugation may be required for certain
antigens). Mice sera will be checked periodically for specific antibodies
by such assay systems as ELISA, Western Blotting, or a specified
assay. Sera will also be made available to the client for further
examination before our proceeding with cell fusion.
2.
Cell Fusion and Screening (clonal selection)
Once the sera is approved by the client, the mice splenocytes are
fused with myeloma cells and clones are screened with ELISA. The
clones that yield a positive ELISA test result will be cultured
(the number of clones kept/cultured will be determined by client
depending on how many monoclonal antibodies are needed). Hybridoma
supernatant will be made available to the client for further testing
if desired.
3.
Cloning
Dilution cloning will be performed on the selected clones to ensure
antibody activity and monoclonality.
4.
Antibody Production
Once antibody activity and monoclonality are ensured, cells will
be injected into mice for ascites production. Ascites will then
be collected and frozen. A sample can be provided for the client
should examination be desired.
5. Antibody Purification
Once ascites collection has terminated, our researchers will determine
the best method of purification to ensure the best combination of
highest purity and activity possible. A report of the proposed purification
strategies and any relevant issues that might arise will be made
available to the client and upon approval, technicians will carry
out the purification. The final product, any frozen ascites/sera,
and cryopreserved cell lines are properties of the client.
.............................................................................................................................................................
|
