Assays
Our in vitro Department at HBI is dedicated to providing a comprehensive suite of in vitro assays designed to support your research and development efforts.
In vitro assays are a critical component of the drug discovery and development process, offering precise, controlled, and reproducible conditions to study cellular and molecular mechanisms.
These assays allow for the rapid screening of compounds, detailed analysis of cellular responses, and elucidation of mechanisms of action, providing essential data to guide the advancement of therapeutic candidates.
Our state-of-the-art laboratories and experienced team ensure that we deliver high-quality, reliable results tailored to meet your specific research needs.
We offer a variety of biocompatibility tests for medical device, established or custom planned assays and validation of assays.
The following methods allowing the evaluation of topically applied compounds, chemicals, cosmetic and personal care product ingredients, and final formulations as well as Medical Devices (ISO 10993-23).
Irritation Studies
Reconstructed Human Epidermis (RHE) – ISO 10993-23, OECD 439
The in vitro Skin Irritation Test is an OECD-approved in vitro non-animal test method for identifying Test Materials that may be irritating to reconstructed human epidermis.
The in vitro skin irritation test is used for the hazard identification of irritant chemicals in accordance with the UN Globally harmonized system of classification and labelling (GHS) Category 2. It aims to predict test compounds potential to cause dermal irritation.
The reconstructed human epidermal tissues consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
The test consists of exposure of the test materials to the reconstructed human epidermis construct tissues followed by a cell viability test using MTT.
MTT assay has been shown to generally be sufficient for predicting irritation, however, various implementations of IL-1α as a secondary endpoint have been used in the industry. These include using IL-1α results to verify borderline MTT assay results or using IL-1α to verify all non-irritant results in the MTT assay.
The results are reported as percent viability (% of living cells).
Test materials are considered non irritant when cells viability is ≥ 50%.
In vitro Eye Irritation Test:
Reconstructed human Cornea-like Epithelium (RhCE) – OECD 492 , ISO 10993-23
The in vitro Eye Irritation Test is an OECD-approved in vitro non-animal test method for identifying Test Materials that may be irritating to reconstructed human corneal epithelium.
The methods is used as an alternative to the traditional In vivo – rabbit eye test method (OECD TG 405).
The test consists of a topical exposure of Test Material onto the Human Corneal Epithelium model HCE, followed by cell viability measurement.
The Reconstructed human cornea-like epithelium is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue and is analogous to the normal in vivo corneal epithelium. The tissue is mitotically and metabolically active and releases many of the pro-inflammatory agents (cytokines) known to be important in ocular irritation and inflammation.
The test consists of exposure of the test materials to the reconstructed human cornea epithelial construct tissues followed by a cell viability test using MTT.
The results are reported as percent viability (% of living cells).
Test materials are considered non irritant when cells viability is ≥ 60%.
HET-CAM assay:
ICCVAM-recommended test
method
The Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) is used to determine the potential irritancy of chemicals, medical devices and materials that are in contact with the eyes or mucous membranes. It can also be suitable for textiles, cosmetics and water.
The HET-CAM is a model that is proposed to mimic the mucosal tissues of the eye and detects vascular injury.
The potential of Test Material to cause irreversible or severe eye irritation or corrosion may be detected by observing adverse changes, which occur in the egg’s CAM after exposure to the test material.
The CAM is a complete tissue containing arteries, veins and capillaries, and is technically easy to study. It responds to injury with an inflammatory process similar to what can be observed in the conjunctival tissue of a rabbit’s eye.
Its well-developed vascularization provides an ideal model for ocular irritation studies.
Test Material is extracted or applied as-is and placed on the egg’s CAM for a few minutes and he blood vessels coagulation, hemorrhage, and lysis are determined.
HET-CAM assay:
ICCVAM-recommended test method
The Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) is used to determine the potential irritancy of chemicals, medical devices and materials that are in contact with the eyes or mucous membranes. It can also be suitable for textiles, cosmetics and water.
The HET-CAM is a model that is proposed to mimic the mucosal tissues of the eye and detects vascular injury.
The potential of Test Material to cause irreversible or severe eye irritation or corrosion may be detected by observing adverse changes, which occur in the egg’s CAM after exposure to the test material.
The CAM is a complete tissue containing arteries, veins and capillaries, and is technically easy to study. It responds to injury with an inflammatory process similar to what can be observed in the conjunctival tissue of a rabbit’s eye.
Its well-developed vascularization provides an ideal model for ocular irritation studies.
Test Material is extracted or applied as-is and placed on the egg’s CAM for a few minutes and he blood vessels coagulation, hemorrhage, and lysis are determined.
Bioassay Method Validation
HBI offers services in designing, developing and validating bioassays.
Our team will help to select the most appropriate assay format and design to achieve a robust and sensitive method before advancing to routine testing.
The routine testing of biologic drugs will ensure your successful drug product manufacturing.
Our team follows GMP guidelines to validate potency assays, focusing on parameters of accuracy, precision, linearity, and specificity. The method validation process is designed to ensure that the assay is reliable and reproducible, providing confidence in the potency results.
USP<1032> GMP guideline -Bioassay Development states the factors to be considered in the design and developing bioassays:
Signal that indicates biological activity,
- Number of doses used in the bioassay
- Dose response
- Determining the method of design and calculation of the bioassay
- Select the reference for relative potency
- Optimizing set points and limits
- Standardizing and automating the bioassay evaluating specificity and stability
USP<1033> GMP guideline -Bioassay Validation states the parameters to be estimated in potency test while monitoring the reference:
- Accuracy
- Precision
- linearity range
- Intermediate precision
- Robustness
- Set point
- Operational range, analyst, day, and instrument variance components
- Stability indicating, significant change in signal under degraded conditions
USP<1034> GMP guideline – Analysis of bioassay data and methods of calculation states that the data analysis of bioassay includes two primary methods for calculation of potency and relative potency:
- 4PL with nine plus doses
- PLA with four doses
In cases where animal life is a consideration, PLA is preferred.
In cases where there is no clear upper asymptote, PLA is required.
Batch Release Testing
GMP Batch Release Testing is conducted for every manufactured drug product batch to ensure each batch is safe and effective for use by patients before it is released to the market.
Biomarkers
Cytokine evaluation:
Detecting and understanding
of the cytokine secretions provides vast information on mechanisms, treatments and the development of new treatments.
Cytokine detection has been used as an inflammatory biomarker for the diagnosis of many diseases.
Cytokines profiles can be characterized in cancer disease, cytokine storm, autoimmune disease, inflammation cytokines, apoptosis etc.
A single cytokine can be detected by ELISA while panels of cytokines can be simultaneously detected by multiplex platforms such as Luminex and flow cytometry.
Mouse Genotyping
Strain name: X
Genomic DNA was purified from mice’s tails. Genotypes were determined according to a real-time PCR melting curves.
Flow Cytometry
The power of flow cytometry is to provide insights into cell populations.
Characterization of various immune cells subtypes by virtue of size and morphology: T cell, B cell, monocyte, NK cell etc.
HBI Services offers bioanalysis using Flow Cytometry for preclinical development and our team help you to establish the most appropriate assay for your research.
Cell based Assays
MTT
XTT
Alamar BlueTM
Monoclonal Antibodies Development and Production
The development of monoclonal antibodies (mAbs) remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies.
The mAbs produced have broad utility as therapeutics, diagnostic tools, and research reagents.
HBI is an expert in the usage of mAbs development technology.
mAbs technology is based on immunization of mice with a specific antigen to prompt a humoral immune response. Antibody-producing B-cells cells from the most immune responder mouse, are fused with a myeloma cell line to generate hybrid cells that are sequentially cloned to obtain stable monoclonal hybridoma.
MAbs resemble natural immunoglobulins from the immunized mouse but differ from serum-derived polyclonal antibodies as they are specific to a single epitope, ensuring a stable, long-term supply.
The MAbs manufacturing process plays an important role in producing therapeutic antibodies widely used in treating diseases such as cancer, autoimmune disorders and other infectious diseases.
HBI offer mAbs production services using bioreactors technology.
The development of monoclonal antibodies (mAbs) remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies.
The mAbs produced have broad utility as therapeutics, diagnostic tools, and research reagents.
HBI is an expert in the usage of mAbs development technology.
mAbs technology is based on immunization of mice with a specific antigen to prompt a humoral immune response. Antibody-producing B-cells cells from the most immune responder mouse, are fused with a myeloma cell line to generate hybrid cells that are sequentially cloned to obtain stable monoclonal hybridoma.
MAbs resemble natural immunoglobulins from the immunized mouse but differ from serum-derived polyclonal antibodies as they are specific to a single epitope, ensuring a stable, long-term supply.
The MAbs manufacturing process plays an important role in producing therapeutic antibodies widely used in treating diseases such as cancer, autoimmune disorders and other infectious diseases.
HBI offer mAbs production services using bioreactors technology.
