Biotechniques BMS 34010A: Immunoassays Quiz
90 Questions
0 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which step is NOT part of the Direct Sandwich ELISA process?

  • Add substrate for colored product development.
  • Coat the ELISA plate with capture antibody.
  • Add a sample containing a target antigen.
  • Incubate the assay without any detection antibody. (correct)

    • Which characteristic distinguishes polyclonal antibodies from monoclonal antibodies?

  • Polyclonal antibodies are produced by a single clone of B-cells.
  • Monoclonal antibodies can recognize multiple epitopes on the same antigen.
  • Monoclonal antibodies are typically used for diagnostic purposes only.
  • Polyclonal antibodies are heterogeneous and derived from multiple B-cell lines. (correct)

    • When performing an agglutination reaction, what is the primary purpose of the antibodies used?

  • To increase the temperature of the reaction environment.
  • To bind to specific antigens and form visible aggregates. (correct)
  • To enhance the permeability of cell membranes.
  • To facilitate the breakdown of cellular components.

    • In an ELISA (Enzyme-Linked Immunosorbent Assay), what is the function of the enzyme linked to the antibody?

    <p>To produce a measurable signal upon substrate conversion.</p> Signup and view all the answers

    Which of the following techniques allows for the simultaneous analysis of multiple parameters in single cells?

    <p>Flow cytometry</p> Signup and view all the answers

    What is the primary advantage of using indirect antibody systems in immunoblotting?

    <p>They are generally cost-effective and offer signal amplification.</p> Signup and view all the answers

    In fluorescence-activated cell sorting (FACS), what property of cells is primarily utilized for sorting?

    <p>Light scattering and fluorescence characteristics</p> Signup and view all the answers

    Which step is NOT part of the Western blotting procedure?

    <p>Cell sorting based on fluorescence.</p> Signup and view all the answers

    What is the role of a blocking solution in immunoblotting?

    <p>To prevent non-specific binding of antibodies to the membrane.</p> Signup and view all the answers

    In indirect immunofluorescence, what is the intermediary molecule that is typically used to detect the bound antigen?

    <p>Secondary antibody</p> Signup and view all the answers

    What is the main reason for protein purification in research?

    <p>To isolate one protein of interest from others for structural and functional studies</p> Signup and view all the answers

    Which mechanism primarily explains the process of ammonium sulfate precipitation?

    <p>Protein solubility decreases as salt concentration increases, leading to precipitation</p> Signup and view all the answers

    What distinguishes the salting out phenomenon from salting in during protein purification?

    <p>Salting out removes water from proteins, while salting in allows proteins to bind to water</p> Signup and view all the answers

    Which of the following best describes the process of protein dialysis?

    <p>It relies on size differences to remove small molecules while conserving larger proteins</p> Signup and view all the answers

    What is the primary mechanism by which SDS-PAGE separates proteins?

    <p>By mass</p> Signup and view all the answers

    Which type of chromatography relies on the ionic charge of proteins for separation?

    <p>Ion exchange chromatography</p> Signup and view all the answers

    What color indicates the presence of proteins in the Lowry protein assay?

    <p>Blue</p> Signup and view all the answers

    During a spectrophotometric measurement, which step is performed before analyzing the sample?

    <p>Conduct a blank measurement</p> Signup and view all the answers

    What technique is utilized to determine the presence of peptide bonds in a substance?

    <p>Biuret test</p> Signup and view all the answers

    What range of wavelengths is measured to quantify the presence of molybdenum blue in the Lowry assay?

    <p>750 nm</p> Signup and view all the answers

    Which of the following techniques is NOT considered as a standard protein purification methods?

    <p>Electrophoresis</p> Signup and view all the answers

    Which statement best differentiates salting in from salting out processes during protein purification?

    <p>Salting in increases protein solubility while salting out decreases it.</p> Signup and view all the answers

    Which of the following statements is true regarding native-PAGE?

    <p>It does not alter protein structure.</p> Signup and view all the answers

    What is the role of the photometer in a spectrophotometer?

    <p>To detect light absorption</p> Signup and view all the answers

    Which chromatography technique relies on the specific binding of proteins to a ligand?

    <p>Affinity chromatography</p> Signup and view all the answers

    Which of the following correctly describes the function of SDS in SDS-PAGE?

    <p>It denatures proteins and provide overall negative charge.</p> Signup and view all the answers

    What is the primary role of the mass analyzer in a mass spectrometer?

    <p>To separate ions based on their mass-to-charge ratios.</p> Signup and view all the answers

    Which factor affects the deflection of ions within the mass analyzer?

    <p>The mass and charge of the ions.</p> Signup and view all the answers

    Why do even molecules that do not typically form ions still work in mass spectrometry?

    <p>They can lose electrons when bombarded by high-energy electrons.</p> Signup and view all the answers

    What characteristic of lighter ions affects their behavior in a magnetic field during mass spectrophotometer analysis?

    <p>They are deflected more than heavier ions.</p> Signup and view all the answers

    Immunoassays measure the presence or concentration of molecules using only antigens.

    <p>False</p> Signup and view all the answers

    Haptens can induce an immune response independently without being attached to a carrier molecule.

    <p>False</p> Signup and view all the answers

    Affinity refers to the overall strength of the antibody-antigen complex.

    <p>False</p> Signup and view all the answers

    Antibodies are Y-shaped molecules made up of only two polypeptide chains.

    <p>False</p> Signup and view all the answers

    Affinity chromatography requires a solvent that is immiscible with the analytes being separated.

    <p>False</p> Signup and view all the answers

    In paper chromatography, the rate at which substances move is dependent on their solubility in the solvent used.

    <p>True</p> Signup and view all the answers

    Column chromatography separates components based solely on their volatility.

    <p>False</p> Signup and view all the answers

    Adsorption chromatography uses a liquid stationary phase to retain analytes on the surface of solid particles.

    <p>False</p> Signup and view all the answers

    Protein purification techniques generally rely on the molecular weight of proteins for their separation.

    <p>False</p> Signup and view all the answers

    The process of salting out refers to the phenomenon where proteins become more soluble at high salt concentrations.

    <p>False</p> Signup and view all the answers

    During protein dialysis, salt molecules move from a more concentrated solution to a less concentrated solution across a semi-permeable membrane.

    <p>True</p> Signup and view all the answers

    Protein gel electrophoresis separates proteins primarily based on their charge and binding affinity.

    <p>False</p> Signup and view all the answers

    Match the following terms with their correct definitions:

    <p>Antibody = Antigen specific proteins that bind to antigens Hapten = Small molecules that require a carrier molecule to induce an immune response Adjuvant = Substances that enhance the immunogenicity of an antigen Affinity = Strength of the interaction between an epitope and an antibody's binding site</p> Signup and view all the answers

    Match the types of immunoglobulins with their primary function:

    <p>IgG = Most abundant antibody in blood; involved in secondary immune response IgA = Found in mucosal areas; protects against pathogens IgM = First antibody produced in response to an antigen IgE = Involved in allergic reactions and responses to parasites</p> Signup and view all the answers

    Match the elements of antibody structure with their descriptions:

    <p>Fab = Fragment responsible for antigen binding Fc = Fragment that is crystallizable Heavy chain = Consists of polypeptide chains in antibodies Light chain = Has a role in the structure of antibodies</p> Signup and view all the answers

    Match the types of immunoassays with their characteristics:

    <p>Direct assay = Detects antigens directly using labeled antibodies Indirect assay = Involves a secondary antibody for detection Sandwich assay = Uses two antibodies to capture and detect the target antigen Competitive assay = Measures the concentration of an antigen by competition with a labeled antigen</p> Signup and view all the answers

    Match the properties of antibodies with their definitions:

    <p>Specificity = Ability of an antibody to bind to one unique epitope Avidity = Overall strength of an antibody-antigen complex Affinity = Strength of interaction between an epitope and an antibody Epitope = Specific part of an antigen that is recognized by an antibody</p> Signup and view all the answers

    Antibodies are Y-shaped protein molecules consisting of four polypeptide chains held together by ______ bonds.

    <p>disulfide</p> Signup and view all the answers

    Haptens are small molecules which could never induce an immune response unless coupled to a ______ molecule.

    <p>carrier</p> Signup and view all the answers

    Immunoassays utilize a label to detect the antigen-______ complex.

    <p>antibody</p> Signup and view all the answers

    Chromatography usually consists of a Mobile Phase and a ______ Phase.

    <p>Stationary</p> Signup and view all the answers

    In chromatography, the time required for a particular analyte to pass through the system is known as the ______ time.

    <p>retention</p> Signup and view all the answers

    The process by which ions, atoms, or molecules adhere to a surface is called ______.

    <p>adsorption</p> Signup and view all the answers

    After ammonium sulfate precipitation, investigators typically apply ______ chromatography procedures to further purify the protein.

    <p>column</p> Signup and view all the answers

    An ______ is a substance analyzed in chromatography.

    <p>analyte</p> Signup and view all the answers

    A ______ is a graphical presentation of detector response used to identify and quantify solutes.

    <p>chromatogram</p> Signup and view all the answers

    In ______ chromatography, the stationary phase is packed into a glass or metal column.

    <p>column</p> Signup and view all the answers

    In paper chromatography, the support material consists of a layer of ______ that is highly saturated with water.

    <p>cellulose</p> Signup and view all the answers

    The ______ coefficient (Kd) describes how an analyte distributes between two immiscible phases.

    <p>partition</p> Signup and view all the answers

    ______ chromatography relies on the ionic charge of proteins for their separation.

    <p>Ion-exchange</p> Signup and view all the answers

    Match the following types of cell cultures with their characteristics:

    <p>Continuous cultures = Can maintain infinite growth Primary cultures = Finite lifespan and limited culture use Immortalized cells = Spontaneous genetic mutation for indefinite growth Adherent cells = Require attachment to a surface for growth</p> Signup and view all the answers

    Match the following cell types with their respective definitions:

    <p>Adherent cells = Anchorage-dependent cells that grow attached Suspension cells = Anchorage-independent cells that grow suspended Heterogeneous cell population = Varied characteristics due to isolation methods Monolayers = Cells growing in a single layer attached to the substrate</p> Signup and view all the answers

    Match the following components of cell culture media with their functions:

    <p>Amino acids = Basic building blocks for protein synthesis Phenol red = pH indicator for monitoring acidity Bicarbonate buffer = Maintains pH in CO2 atmosphere Antibiotics = Prevents contamination by microorganisms</p> Signup and view all the answers

    Match the disadvantages of cell culture components with their implications:

    <p>Fetal Calf Serum = Risk of infectious agents like prions Primary cultures = Limited lifespan affects experimental duration Immortalized cells = Loss of original in vivo characteristics Suspension culture = Can lead to easier cross-contamination</p> Signup and view all the answers

    Match the following terms related to cell culture with their descriptions:

    <p>Growth factors = Enhance cell attachment and proliferation Inorganic salts = Help maintain osmotic balance in culture media Growth curve lag phase = Period of active cellular processes before visible growth Plasmids = Genetic elements used for transformation in immortalization</p> Signup and view all the answers

    What distinguishes the affinity from the avidity of antibodies?

    <p>Affinity is the strength of binding between a single epitope and an antibody, while avidity refers to the overall strength of the antibody-antigen complex.</p> Signup and view all the answers

    Define proteomics and its relevance in the study of proteins.

    <p>Proteomics is the large-scale study of proteins, particularly their functions and structure, providing insights into cellular processes and protein interactions.</p> Signup and view all the answers

    What two phases are integral to the process of chromatography, and how do they function?

    <p>The two phases are the mobile phase, which carries the analyte, and the stationary phase, which the analyte interacts with during separation.</p> Signup and view all the answers

    What role does retention time play in chromatography, and why is it important?

    <p>Retention time is the duration an analyte takes to pass through the chromatography system, which is important for identifying and quantifying substances in a mixture.</p> Signup and view all the answers

    What differentiates affinity chromatography from other forms of chromatography?

    <p>Affinity chromatography utilizes specific interactions between an analyte and a ligand attached to the stationary phase, allowing for selective separation based on biochemical properties.</p> Signup and view all the answers

    Which component is NOT part of a mass spectrometer?

    <p>Spectral detector</p> Signup and view all the answers

    What is the first step in the process of mass spectrometry?

    <p>Ionization</p> Signup and view all the answers

    In mass spectrometry, what happens to ions subjected to deflection?

    <p>They curve away from their original path</p> Signup and view all the answers

    Which of the following best describes what occurs during the ionization step in mass spectrometry?

    <p>Molecules are bombarded with electrons</p> Signup and view all the answers

    What causes different masses in isotopes of the same chemical element?

    <p>Different number of neutrons</p> Signup and view all the answers

    What is a primary application of mass spectrometry for environmental monitoring?

    <p>Analyzing soil and water pollutants</p> Signup and view all the answers

    In the context of drug testing, what is a role of mass spectrometry?

    <p>To identify drug abuse and metabolites</p> Signup and view all the answers

    Why do molecules that typically do not form ions still work in mass spectrometry?

    <p>They can be ionized under certain conditions</p> Signup and view all the answers

    What is the role of the vacuum pump in a mass spectrometer?

    <p>To remove unwanted gaseous ions from the system</p> Signup and view all the answers

    What is the main role of adjuvants in immunology?

    <p>To enhance the immunogenicity of antigens</p> Signup and view all the answers

    Which class of immunoglobulin primarily exists in the extracellular fluid and accounts for 70-75% of all human immunoglobulins?

    <p>IgG</p> Signup and view all the answers

    What makes antibodies highly specific in their function?

    <p>The unique antigen binding site at the tip of their variable region</p> Signup and view all the answers

    What is the definition of avidity in the context of antibodies?

    <p>The overall strength of multiple binding interactions between antibodies and antigens</p> Signup and view all the answers

    Which statement correctly describes the structure of antibodies?

    <p>Antibodies are Y-shaped proteins made up of four polypeptide chains</p> Signup and view all the answers

    What is necessary for agglutination to occur?

    <p>A specific ratio of antibody to antigen.</p> Signup and view all the answers

    What is the role of the capture antibody in a Direct Sandwich ELISA?

    <p>It coats the ELISA plate to capture the target antigen.</p> Signup and view all the answers

    What does immunohistochemistry primarily visualize?

    <p>The localization of proteins and other macromolecules in tissues.</p> Signup and view all the answers

    Which of the following is necessary to visualize the enzyme linked antibody in an ELISA?

    <p>Colored substrate</p> Signup and view all the answers

    What type of signal does a Fluorescent ImmunoAssay utilize?

    <p>Fluorescent dyes</p> Signup and view all the answers

    In the context of immunoassays, what does the term 'sensitivity' refer to?

    <p>The capacity to detect low levels of antibody or antigen.</p> Signup and view all the answers

    Study Notes

    Biotechniques (BMS 34010A)

    • Course offered Fall semester 2023-2024
    • Instructor: Dr. Tania Tahtouh
    • Email: [email protected]

    Immunoassays

    • Immunoassays are biochemical tests for detecting or quantifying molecules using antibodies or antigens.
    • The principle relies on the specificity of antibody-antigen reactions.
    • Various formats exist, all involving antibody binding to the target antigen and a detectable label.

    Definitions

    • Antigen (Ag): A substance triggering an immune response, serving as targets for antibodies.
    • Antibody (Ab) or Immunoglobulin (Ig): Antigen-specific proteins; binding is specific to the initiating antigen.
    • Different classes exist: IgA, IgD, IgE, IgG, and IgM.
    • Haptens: Small molecules needing a carrier protein for immunogenicity.
    • Adjuvant: Substances enhancing antigen immunogenicity and reducing needed antigen amounts.

    Immunoglobulin Classes

    • IgG is the most prevalent immunoglobulin in the extracellular fluid, accounting for 70-75% of total immunoglobulins in human blood.
    • IgG's small size and high diffusibility make it prevalent in the extracellular fluid.

    Antibody Structure

    • Antibodies are Y-shaped protein molecules with four polypeptide chains held together by disulfide bonds.
    • Two types of chains exist: heavy and light chains.
    • Two types of domains are variable and constant
    • Two parts of the molecule are Fab (fragment antigen-binding) and Fc (fragment crystallizable).

    Antibody Properties

    • Specificity: Unique antibody binding to a particular epitope.
    • Affinity: Strength of interaction between an epitope and antibody's antigen-binding site
    • Avidity: Overall strength of an antibody-antigen complex.

    Polyclonal Antibodies

    • A complex mixture of antibodies recognizing multiple epitopes of a single antigen.
    • Obtained from various animal sources (e.g., rabbits).

    Monoclonal Antibodies

    • Laboratory-produced antibodies targeting a specific antigen.
    • Produced from a single clone of plasma B cells.

    Agglutination

    • A visible reaction resulting from antibody-antigen aggregation.
    • Optimum agglutination depends on the right ratio of antibodies and antigens.
    • Significant in blood typing and other immunological diagnostic tests.

    Agglutination Types

    • Different reactions (e.g., antibody or antigen excess) cause visible or non-visible agglutination.

    Immunoassays by Signal

    • ELISA (Enzyme-Linked Immunosorbent Assay): Uses colored substrate measured by absorbance.
    • FIA (Fluorescent ImmunoAssay): Uses fluorescent dyes measured by a microplate fluorometer.
    • RIAS (Radio Immunoassay): Measurable signal from radioisotopes, often using gamma counters.

    ELISA Types

    • Direct ELISA: Direct detection of antigen with a labeled primary antibody.
    • Indirect ELISA: Indirect detection by reaction of labeled secondary antibody binding to a primary antibody, which binds to the antigen.
    • Sandwich ELISA: Capture antibody immobilizes antigen.
    • Competitive ELISA: Compete for limited binding sites on the capture molecule; reduce signal if antigen is present in the sample.

    Direct Sandwich ELISA

    • Coats ELISA plate with capture antibody
    • Adds target antigen (sample)
    • Adds enzyme-labeled detection antibody
    • Substrate for colorimetric detection, and measuring via a standard curve

    Immunohistochemistry (IHC)

    • Powerful microscopy for visualizing cellular components, proteins within tissue samples.
    • Immunofluorescence (IF): Uses antibodies tagged with fluorescent markers which are then visually confirmed via UV illumination.

    Common Fluorophores for Ab Conjugation

    • List of fluorophores for antibody conjugation with associated excitation wavelengths

    Immunoblotting (Western Blotting)

    • Technique for detecting specific proteins from electrophoretic sample separation.
    • Methods for blotting and detecting protein specificity.

    Flow Cytometry

    • Laser-based technology for analyzing cells in suspension (simultaneous measures); physical and chemical characteristics.
    • Used for analyzing cell-surface and intracellular molecules, characterizing cell types and purity of isolated cell populations, and analyzing cell size.

    Fluorescent Activated Cell Sorting (FACS)

    • Specialized flow cytometry for sorting cells based on light scattering or fluorescence.
    • Sorts cells based on their light scattering and fluorescent characteristics separately to multiple containers.

    Autofluorescence

    • Fluorescence from naturally occurring compounds (e.g., chlorophyll in plant tissue, collagen).
    • Often observed at specific wavelengths, with excitation commonly occurring near the UV region (~365 nm).

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Related Documents

    Immunoassays PDF Fall 2023-2024
    Protein Structure & Purification PDF
    Mass Spectrometry PDF
    Chromatographic Techniques PDF
    Lecture 6 - Cell Culture PDF

    Description

    This quiz covers key concepts in immunoassays, focusing on the role of antibodies and antigens in biochemical tests. Students will explore immunoglobulin classes, definitions related to immune responses, and the various formats of immunoassays. Prepare to test your understanding of these essential biotechnology principles.

    More Like This

    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser