BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

                  MCQ on analytical methods used for for microbial identification 1. Which type of test involves analyzing the metabolic properties of microorganisms for identification? a) Serological tests b) Nucleic acid sequencing c) Biochemical tests d) MALDI-TOF mass spectrometry 2. Which of the following test uses antibodies to detect specific antigens on the surface of microorganisms for identification? a) Enzyme-linked immunosorbent assays (ELISA)      b) Phage typing c) Polymerase chain reaction (PCR) d) Whole Genome Sequencing (WGS) 3. Which analytical technique involves the amplification of specific DNA regions for detecting unique DNA sequences of microorganisms? a) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) b) Polymerase Chain Reaction (PCR)     c) Phage typing d) Serological tests 4. Which analytical technique targets a conserved region of the ba...

Bioanalytical Methods to Improve Drug Tolerance 1. Acid Dissociation 2. Solid-phase extraction with acid dissociation (SPEAD) 3.  Affinity Capture Elution (ACE) 4. Biotin-drug extraction and acid dissociation (BEAD) 5. Precipitation and acid dissociation (PandA) 1. Acid Dissociation methods: Acid-base treatment is a widely adopted dissociation method for improving the drug tolerance of the assay. The method is based on the principle that acid or base breaks any drug: antibody complex interaction thereby allowing the detection/capture reagents to the ADAs.  The method involves incubation of ADA: drug complex with weak acid with pH 2-3 (e.g 300mM acetic acid, 0.1 mM Glycine) followed by neutralization with base (1 mM Tris HCl, pH 9.5), whereas if a high concentration of circulating drugs is present, there is a higher probability that ADA re-associates with the drug during the neutralization step. The acid may also negatively affect the assay signal/noise ratio ...

 To label mitochondria, cells are simply incubated with commercially available Mito Tracker® fluorescent dye such as tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE), Rhodamine 123 which passively diffuse across the plasma membrane and accumulate in active mitochondria. Once their mitochondria are labeled, the cells can be treated with an aldehyde-based fixative for samples that need fixation to allow further processing of the sample. Some of the Mito Tracker® probes are also retained after permeabilization with some detergents during subsequent processing steps (e.g., immunocytochemistry or in situ hybridization). MitoTracker Red CMXRos MitoTracker Red CMXRos is a red-fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon membrane potential. The dye is well-retained after aldehyde fixation. allowing for further sample processing and immunostaining. Excitation: 579 nm, Emission: 599 nm,  Invitroge...

       Acid capture elution (ACE) is a plate-based method for reducing the drug interference in the ADA (Adenosine deaminase) assay. It is used for the purification and isolation of specific nucleic acids, such as DNA or RNA, from a complex mixture. This method utilizes weak organic acids (Glycine, Acetic acid) to dissociate the drug-ADA complex.  The sample is neutralized and allowed the bind the plate coated with the drug. After the ADA is captured, the plate is washed to remove any unbound drug present in the sample.  The second acid treatment is used to elute the ADA bound to the plate.  The ADA sample is transferred, neutralized with Tris-HCl base, and processed in the ADA detection assay.  Procedure: 1) Binding (Coating of the plate with a drug): Maxisorp ELISA Nunc plate is widely used for coating the plate that would be used for the capture of ADA. For the optimization of drug concentration, test different concentrations of drug (0....

History of Gene Therapy, Vector DNA integration, and Safety concern: After more than two decades of setbacks and scrutiny, gene therapy has reemerged in the mainstream of drug discovery. This has renewed hope and enthusiasm among patients, healthcare providers, and drug developers.  During early clinical trials, there were incidences of life-threatening safety risks associated with gene therapy and death, these events brought the human trials near to a halt.  Genotoxicity, cancer induction, and progression were common safety events observed in numerous clinical trials.   It was believed that these gene therapy products may have induced  activation of protooncogene, clonal expansion of these tumorigenic cells, and progression of leukemia. The viral vector DNA insertion into the host DNA may be associated with the mutation resulting in various tumor-promoting phenomena. Viral Vectors, Gene Integration, and Integration Assay: Lentivirus , Gamma retroviruses , a...

The circulating drug binds to the anti-drug antibodies (ADA) in the samples and interferes with the detection of ADA. The circulating drug severely reduces the sensitivity of the assay and increases the false-negative rate during clinical bioanalysis.  Drugs can interfere with ADA detection by two mechanisms;  a) Circulating drugs form immune complexes with ADA b) Circulating drugs compete with the detection reagents in the assay  Biotin-Drug Extraction and Acid Dissociation (BEAD)  procedure: BEAD method has shown to effectively remove the circulating drugs from the serum samples and reduce the drug interference in the assay. The BEAD method utilizes acetic acid to dissociate the drug-ADA complex. The sample is rapidly neutralized in the Tris-HCl containing an excess of biotinylated drug and allow binding to ADA to biotinylated drugs.  The streptavidin-coated magnetic Bead is used to capture the biotinylated drug-ADA complex and remove any free circul...

FDA guidance for Immunogenicity Testing Assays:   Development & Validation of Anti-drug Antibody Assays The FDA has finalized its guidance for the development and validation of assays for immunogenicity testing. The new guidance supersedes other previous guidance for related to bioanalytical assay validation for immunogenicity testing. The objective of this guidance is to facilitate the industry’s development and validation of assays for assessment of the immunogenicity of therapeutic protein products during clinical trials. More specifically the guidance provides a stepwise approach to develop, validate the anti-drug antibody assay, it helps to answer the following questions: a) Why it is important to assess immunogenicity? b) What is the ADA assay? c) When is it appropriate to develop ADA assays and assess immunogenicity? d) How to develop reliable, sensitive and specific ADA assays? a) Why it is important to assess immunogenicity? Immunogenicity is defined a...

Introduction to Anti Drug-Antibody Assay The total anti-drug antibody(TAb) assays detect the antibodies that are specific to the therapeutic molecules. These assays do not distinguish specific isotypes and detect all classes of antibodies present in the samples. The different platforms have been used for the detection of total antibodies such as bridging antibody assays, indirect ELISA assays, SPR assays, Immuno-PCR assay and most recently mass spectrometric assay.  Although the detection system varies with different platforms, the detection still relies on the ADA and therapeutic molecules binding and interactions. A tier-based approach is utilized for detection of ADA that includes a) Screening Assay b) Confirmation Assay c) Titer Assay a) Screening Assay : The screening assay is designed to detect and distinguish the presence or absence of anti-drug antibodies in the samples. The screening assays involve the incubation of samples with capture and detection reagents. Th...

Anti-drug antibody (ADA) assays are developed to detect and characterize an antibody response generated against bio-therapeutic products. The total antibody (TAb) assay (TAb) detects the total antibodies generated against the therapeutic proteins. The total antibody assay positive samples that are positive for total ADA are tested for the neutralizing capacity. These TAb-positive samples may be subjected to isotype characterization as necessary. These assays are performed in three tiers : a) screening assay, b) confirmation assay, c) titer assay  Assay cut points are established with three basic assumptions The cut points are usually a statistically determined cut off value above which the samples are considered positive. During pre-study validation, the cut points are established by analyzing a set of approximately fifty drug-naïve healthy individual samples. The cut points are established with the following basic assumptions: a) No prior exposure to the drug b) No preexisting...

 Bead Extraction and Heat Dissociation (BEHD) For Mitigation of Drug Interference BEHD is the method developed for mitigating drug interference. BEHD method utilizes heat denaturation for the dissociation of the ADA-drug complex present in the samples. The samples are subsequently incubated with the sample and ADA is allowed to bind with Biotin-Drug and captured using streptavidin coated magnetic beads.  Next, the samples are eluted using weak acids such as acetic acid or glycine, neutralized, and assayed in the ADA or NAb assay. The heat denaturation steps become critical when the drugs that are used to capture ADAs are susceptible to harsher acid-base conditions. This BEHD is used for the detection of ADA for cytokine therapy.  Protocol For BEHD : i) Heat Denaturation for Dissociation for ADA-Drug Complex 50ul to 100 ul of the serum samples containing ADAs are treated with mild heat at 62 degrees Celsius for 1 hour in thermocycler and allow the dissociation of ...

            Solid Phase with Extraction  Acid Dissociation (SPEAD) is a plate-based method for reducing drug interference in the ADA assay. This method utilizes weak organic acids (Glycine, Acetic acid) to dissociate the drug-ADA complex. The sample is neutralized and allowed the bind to a biotinylated drug and captured on the streptavidin-coated 96 well plate.  After the ADA is captured, the plate is washed to remove any unbound drug present in the sample. The second acid treatment is used to elute the ADA bound to the plate. The ADA sample is transferred, neutralized with Tris-HCl base, and processed in the ADA detection assay.  Procedure: 1) Coating of the plate with a drug: Streptavidin-precoated 96 well high binding plate is widely used for capturing the biotinylated drug / ADA complex. For the optimization of drug concentration,  Test different concentrations of biotinylated drug ( 0.5, 1.0, 5.0 ug / ml, and 10 ug/ml ) and ch...

          Bispecific antibodies consist of two small-chain fragments variable (scFv) of the antibody that has binding affinities to different target antigens. T cell engagers are a class of bispecific antibodies that selectively recruit and activate T cytotoxic cells in the tumor tissue and mediate cytotoxicity. In T cell engagers, one scFv domain is designed to bind to CD3 molecules in the T cell, and the second scFv domain binds to a tumor-associated antigen expressed in the tumor tissue. Certain tumor-associated antigens are known to express in specific cancer types. These tumor-associated antigens have been targeted for the treatment of these specific cancers.  These tumor antigens include CD19, BCMA, PSMA, HER2, TROP2, etc.            Bispecific antibodies, similar to any protein therapeutics, can evoke the generation of anti-drug antibodies. Therefore, detection and monitoring of anti-drug antibodies is an i...

S urface Plasmon Resonance Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real-time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions. SPR signal is directly dependent on the refractive index of the medium on the sensor chip. The binding of biomolecules results in changes in the refractive index on the sensor surface. In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to a second molecule (the Analyte) is measured under flow. Response is measured in resonance units (RU) and is proportional to the mass on the surface, and for any given interactant, the response is proportional to the number of molecules bound to the surface. Response is recorded and displayed as a sensogram in real time. Principle of Surface Plasmon Resonance (GE) Application of SPR Increase understanding of molecular mechanisms and structure-function relationships Define poten...

Ion exchange chromatography Principle:  Ion exchange chromatography is a technique for separating and purifying the compounds in the mixture based on their net charge. Ion exchange chromatography resin contains negatively or positively charged functional groups covalently bound to a solid support, yielding either a cation or anion exchanger, respectively.  Charged compounds are adsorbed and retained by an ion exchanger having the opposite charge, whereas compounds that are neutral or have the same charge as the media pass through the void volume and are eluted from the column.  The binding of the charged compounds is reversible, and adsorbed compounds are commonly eluted with a salt or pH gradient. Ion exchange media are available in various particle sizes, ionic forms, and purity ranges.  Ion exchange chromatography is used both for preparative and analytical purposes and can separate a large range of molecules from amino acids and nucleotides to large prote...