BIOASSAY DEVELOPMENT

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BIOASSAY DEVELOPMENT : 

BIOASSAY DEVELOPMENT BY PRAVESH VERMA M.PHARM[PHARMACOLOGY] 2nd SEM BABASAHEB BHIMRAO AMBEDKAR UNIVERSITY,LUCKNOW,UP,INDIA

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DEFINITION: Bioassay development is the process to obtain a final assay system that is appropriate for its intended use and can be reliably performed repeatedly A bioassay is defined as an assay that measures biological activity of a substance based on the response of a biological test system to the test substance The final bioassay protocol is developed through careful evaluation of all potential parameters that may affect the assay BIOASSAY DEVELOPMENT

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In the pharmaceutical industry, bioassays are commonly applied to characterize a substance’s biological properties, to study a biological process, to detect the presence and quantity of a substance in a sample, and to screen for active molecules from a library of molecules. AIM

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Before a substance is approved for human Use:- it has to be fully characterized. The characterization of a substance requires the determination of its physiochemical properties by physiochemical assays (characterization) And the determination of its biological activities by bioassays. NEED

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a substance’s biological activity cannot be measured by studying the drug substance alone A biological test system is required for a bioassay REQUIREMENT OF BIOASSAY

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The biological test system can be :- (1) Biochemical :- such as the activity of an enzyme or the ability to bind to a predefined protein (2) cell based :- such as isolated primary cells or transformed cell lines (3) tissue or organ based. (4) animal based. BIOLOGICAL TEST SYSTEM

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Due to the diversity of the choices of the biological test system :- A variety of a bioassay can be developed for a given project The most common bioassays are :- 1.Biochemical assays with isolated proteins 2.Cell-based assays.

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Bioassay development is the process to obtain a final assay system that is appropriate for its intended use and can be reliably performed repeatedly The final bioassay protocol is developed through careful evaluation of all potential parameters that may affect the assay BIOASSAY DEVELOPMENT

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The bioassay development process can be divided into following parts :- A. Carefully studying the biological target to determine what biological parameters should be determined to answer specific questions

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C.Understanding the selected measurement’s application range (boundary conditions) and making sure the intended uses are within the boundary D. Obtaining a control substance known to generate the intended responses in the test system E. Building algorithms that can mathematically relate the final detected signal to the intrinsic biological function

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A growth factor (L) is a potential therapeutics found in drug discovery phase and it is currently under development. A growth factor (L) is a potential therapeutics found in drug discovery phase and it is currently under development. It is known that L may denature in some formulation resulting in lose of activity Thus an assay for L is needed to support the preclinical testing in mice. EXAMPLE OF BIOASSAY DEVELOPMAENT

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PROCESS :- A. Define the Goal of the Assay:- The goal here is to determine the active substance in each batch of L to guide the administration of correct amount of L into mouse. B. Design an Assay:- It is known that L binds to a receptor (R) to elicit the downstream biological effects that form the basis for the intended therapeutic use of L. Thus, a biological test system that contains receptor R can be constructed.

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The response from the test system can be directly measured when L binds to receptor R. It is also known that L’s biological activity started with its binding to the cell surface receptor R. The binding of L initiated the dimerization of receptor R on the cell surface. This is followed by autophosphorylation of receptor R that is followed by many other biological responses From this information, a test system based on cells that express receptor R at their surface can be established.

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With this test system, the biological activity of L can be measured by the binding between L and R, the dimerization of R, the phosphorylation of R, or other downstream biological responses. After detailed analysis with these different readouts, the phosphorylation of R is chosen as the final assay readout.

Figure :- Illustration of bioassay for active ligand (L) of a growth factor receptor (R).1. The first biological test system (System 1) is a cell line that expresses the receptor on its surface.2. The second biological test system (System 2) is an ELISA system with the first antibody (Abl) attached to a solid surface that captures solubilized receptor R.3. The fluorescence-labelled second antibody (Ab2) binds only to phosphorylated R.4. Upon removal of the unbound Ab2, the fluorescent signal can be measured with a fluorimeter.5. The intensity of the fluorescence is proportional to the quantity of the phosphorylated receptor R that in turn is proportional to the quantity of the active ligand L. : 

Figure :- Illustration of bioassay for active ligand (L) of a growth factor receptor (R).1. The first biological test system (System 1) is a cell line that expresses the receptor on its surface.2. The second biological test system (System 2) is an ELISA system with the first antibody (Abl) attached to a solid surface that captures solubilized receptor R.3. The fluorescence-labelled second antibody (Ab2) binds only to phosphorylated R.4. Upon removal of the unbound Ab2, the fluorescent signal can be measured with a fluorimeter.5. The intensity of the fluorescence is proportional to the quantity of the phosphorylated receptor R that in turn is proportional to the quantity of the active ligand L.

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C. Understanding the Measurement :- There are many bioassays to measure the phosphorylation state of membrane receptors Here we choose to measure the phosphorylation of receptor R with a sandwiched enzyme-linked immunosorbent assay (ELISA) system. In this assay, one antibody attached to the bottomsurface of a microplate captures solubilized receptor R. Another fluorescently labelled antibody recognizing the phosphate group is used for detection.

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D. Obtaining a Control Substance Known to Elicit the Desired Response from the Test Systems:- The proposed assay contains two consecutive assays with two test systems. The first test system is the cell line that expresses receptor R and is responsive to active L. The second test system is the ELISA, which is responsive to the phosphorylated R.

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E. Relating Measurement to Intrinsic Biological Function :- The raw data obtained from the above bioassay is the relative fluorescence unit (RFU). the RFU is converted to units of control ligand L based on the response generated

PERFORMANCE CHARECTERISTICS OF ASSAY DEVELOPMENT PROCESS : 

PERFORMANCE CHARECTERISTICS OF ASSAY DEVELOPMENT PROCESS Accuracy 2. Precision 3. Sensitivity 4. Specificity 5. Detection Limits 6. Lower Quantitation Limits 7. Upper Quantitation Limits 8. Linearity

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9. Dynamic Range 10. Robustness 11. Boundary Conditions 12. Reproducibility 13. Scalability

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