In Vitro Services

The In Vitro Services department at Biotox Sciences provides clinical pathology services for all animal species, bioanalytical for biologics and cell-based functional assays to support pharmaceutical product development.
Biotox Sciences offers its clients several in vitro assays to help determine the efficacy, mechanism of action and toxicity of their compounds. We have over 350 cancer cell lines and primary normal cells and various drug resistant cell lines, valuable tools to evaluate small drug molecules and Biologics, siRNA and liposomal formulated drugs.

Target Exclusivity
To maximize the value to clients and not enter into conflicts of interests, BTS screens on a target exclusive basis. This means that each and every assay can be an opportunity for a new discovery that may be unavailable to subsequent clients. The availability of the proposed target is evaluated confidentially at an early stage of discussions.

Detailed Research Plan
Before starting a partnership, BTS will develop a detailed research plan with the scientists of the potential partner. The plan will be developed once a business agreement is reached with the client. The comprehensive research plan will describe the assay, its development, and the goals of the Lead Compound Discovery project.

Analytical Testing

BioTox Sciences conducts a variety of tests in-house in addition to their time tested collaborating partners for small molecules bioanalysis. Our in-house analytical services utilize high through put equipment which is recognized as an industry benchmark. The following tests are conducted in house.
RT-PCR PCR Q-PCR Clinical chemistries Hematology	Coagulation Blood gas ELISA Cytokine analysis FACS

Antibody Production & Characterization +

Our Custom Monoclonal Antibody Service Gives You:

Immunization of 5 Balb/c mice.
Fusion of spleen cells with SP2/0 myeloma cells.
ELISA screening for selection of antibody producers.
Subcloning and expansion of selected antibody producers.

Breakdown of Standard Monoclonal Service by Phase
Phase I 6 Weeks	• Immunization and boosts of 5 Balb/c mice with standard immunization protocol. • ELISA evaluation of titer prior to selection for fusion. • Approximately 3-4 mg protein immunogen or 2 mg conjugated peptide immunogen and 0.5 mg free peptide are required.
Phase II 4-6 Weeks	• Once an acceptable titer is obtained, hybridoma fusion will be done using splenocytes from mouse with the best titer and myeloma cells. • Supernatants from the growing hybridoma wells will be screened by ELISA. If needed, 10 clones will be sent to customer's lab for evaluation
Phase III 4-8 Weeks	2 of these 10 clones selected by the customer will be subcloned by limiting dilution, isotyped, and expanded for cryopreservation.

Our Custom Polyclonal Antibody Service Gives You:

	Service	Description	Time
1	Immunization and anti-sera production	• Conjugated peptides or recombinant proteins are used to immunize rabbits. • Ten-week protocol to produce antibodies in 2 rabbits. Pre-immune and three immune bleeds (100-120 ml antiserum).	7-10 weeks
2	Test	• ELISA test until the titer reaches 1:4,000.	1 day
3	Purification	• Protein G purification • Antibodies are eluted using both pH11 and pH2.5 buffers and neutralized immediately followed by dialysis in PBS.	1 week
	Total		10-13 weeks

In Vitro Functional Assays +

Assay Development
Like any new project, additional work might be needed to prepare the assay(s) for high-throughput screening. Assay development is defined broadly, from simple technology transfer to complete de novo development depending on the client's target and the assay's stage of development. BTS will apply the best technologies for measuring and screening of biomolecular interactions and functions of cells to reduce the expense and to improve the efficiency of the screening process. For example, the use of the 1536 microplates will reduce the cost and increase the speed of screening.

Services

Cell viability (cytotoxicity)
Cell proliferation
Cell cycle analysis
Apoptosis
Cell adhesion
Cell migration
Cell invasion
Chemotaxis
Angiogenesis
Soft Agar Colony Formation Assay
Cell culture and expansion of various primary and cancer cells
Receptor binding
Calcium Flux
Intracellular pH change
Cell shape change
Endotoxin measurement (LAL assay)
Mechanism of action, intracellular signaling pathway identification
Cell differential count of lung infiltrating cells in asthma studies by cell cytospin staining and microscopic evaluation
Mast cell isolation and histamine release in vitro
Drug degradation ex vivo in whole blood
Heparin compatibility drug evaluation
Ex vivo antibody production
Oxygen/glucose deprivation: evaluation of anti-apoptotic laser treatment in rat CNS cell culture
Cellular uptake of FITC-labeled drug

Antibody production and characterization
Dendritic cell vaccine for cancer models treatment
Primary Cell Function Assays

Neutrophils Functions: degranulation, oxidative burst, calcium mobilization, adhesion, chemotaxis, cytokines and lipid mediators production, integrin upregulation
Macrophage Function: Degranulation, phagocytosis, chemotaxis, calcium mobilization, integrin upregulation
T Cell Function: proliferation, cytokine production, chemotaxis, T-B cognate interaction, MLR, calcium mobilization, phosphorylation, TH1/Th2/Th3/Treg assays, antigen T cell recall.
B Cell function: Ig production, Ig Switching, chemotaxis, calcium mobilization, proliferation, phosphorylation
Dendritic Cell Function: maturation, antigen presentation, cytokine production, chemotaxis, calcium mobilization, phosphorylation
NK Cell Function: cytolytic activity, ADCC, CDC, chemotaxis, calcium mobilization, phosphorylation, cytokine production
Endothelial Cells: tube formation, cell-cell interaction, cell-substrate interaction, chemotaxis, migration, integrin up-regulation
Hepatocytes: proliferation, toxicity, drug metabolism
Sebocytes: cell proliferation, sebum production
Keratinocytes: proliferation, chemotaxis, cytokine production, phosphorylation
Cardiac toxicity: drug induced toxicity is evaluated in vitro on 16 primary heart cell types, release of LDH is measured at 24 and 48 hr post treatment.
Platelet activation, upregulation of markers of activation
RBC lysis Assay

Custom in vitro assays

Biomarkers +

Biomarker identification and validation
Flow cytometry (cell surface and intracellular staining)
Western blot
Zymogels for protease activity
ELISA (development, validation and use of commercial kits)
Multiplex technology for cytokines
Pharmacodynamic studies: quantification of drug target inhibition following animal treatment
Factor VII A measurement in dogs and mice
HSV2 and Clamydia measurement by Q-RTPCR
Nitrite/nitrate measurements
Butyrylcholinesterase and Carboxylesterase-1 and -2 by Western Blot
Beta amyloid measurement by ELISA
Hydroxyproline measurement to evaluate anti-liver fibrosis drug
Ds DNA IgG measurements by ELISA