In the enzyme-linked antibody used in ELISA, the interaction between the enzyme and antibody is stabilized by
covalent bond
ELISA Principle and Enzyme-Antibody Linkage
ELISA, which stands for Enzyme-Linked Immunosorbent Assay, is a widely used technique in biology and medicine. It is primarily used to detect the presence of an antigen or an antibody in a sample. A key component in many ELISA formats is an antibody that is chemically linked, or conjugated, to an enzyme. This enzyme provides a detectable signal when a specific substrate is added, indicating the presence of the target molecule (antigen or antibody).
Enzyme-Linked Antibody Function
For the ELISA assay to work reliably, the link between the enzyme and the antibody must be stable and permanent. The enzyme needs to remain attached to the antibody throughout the assay steps, including washes, to ensure that the signal generated is directly related to the binding event between the antibody and the target molecule.
Bonding Types in Molecular Interactions
Let's consider the types of interactions mentioned in the options:
- Hydrogen bonds: These are relatively weak, temporary interactions formed between molecules containing hydrogen bonded to a highly electronegative atom (like oxygen or nitrogen) and another electronegative atom. They are common in biological systems (like in water or DNA structure) but are generally not strong enough to create a permanent link required for enzyme-antibody conjugation.
- Ionic bonds: These bonds form between oppositely charged ions. While stronger than hydrogen bonds, they can be disrupted by changes in salt concentration or pH, making them less suitable for a stable linkage in an assay involving various buffer conditions.
- Van der Waals interactions: These are very weak, short-range forces that arise from temporary fluctuations in electron distribution around atoms. They are not suitable for creating stable chemical bonds between molecules like an enzyme and an antibody.
- Covalent bonds: These bonds involve the sharing of electron pairs between atoms. Covalent bonds are strong and stable chemical bonds. They require specific chemical reactions to form and are not easily broken under typical biological or assay conditions (like different buffers or washes).
Covalent Bond for Stable Linkage
In the enzyme-linked antibody used in ELISA, the enzyme is chemically conjugated to the antibody. This conjugation process involves forming a stable chemical bond between specific functional groups on the enzyme and the antibody molecules. The type of bond formed in this chemical linking process is a covalent bond.
Using covalent bonds ensures that the enzyme remains permanently attached to the antibody throughout the assay procedure. This stable linkage is crucial for the accuracy and reliability of the ELISA results, as it guarantees that the enzymatic activity detected corresponds directly to the binding event of the antibody to its target.
Therefore, the interaction (linkage) between the enzyme and antibody in the enzyme-linked antibody used in ELISA is stabilized by a covalent bond.
Important Questions from Histochemical and Immunotechniques
For a given immunological application [column X], select the type of antibody [column Y] that should be used:
Choose the option with correct matches between terms of Columns X and Y.Column X Column Y A Bacterial agglutination (i) Only monoclonal B Western blotting (ii) Only polyclonal C Detection of a cytokine using a solid phase ELISA (iii) Either monoclonal or
polyclonalD Diagnostic tissue typing A researcher raised antibodies against sheep red blood cells (SRBCs) and purified the IgG fraction. Some of the IgG antibodies were then subjected to enzymatic digestion to have Fab, Fc and F(ab’)2 fractions. He placed each preparation in a separate tube ( 1 to 3), labeled the three tubes to indicate their contents, and incubated them on ice. After a while he noticed that the label on two of the tubes (1 and 2) had gotten erased. He did a test for tube 1 and found that the preparation in the tube agglutinated SRBCs but did not lyse them in presence of complement. Which preparation was in tube 1?
To investigate the relationship between microtubules and centrioles in fixed HeLa cells using an epifluorescence microscope, a researcher plans to conduct immunostaining using antibodies against tubulin and centrin (centriolar protein). After the incubation with the primary antibodies and wash, she/he plans to use secondary antibodies that bind to the primary antibodies. Below is a list of secondary antibodies carrying various fluorophores (dyes) available to the researcher.
A. Alexa 568
B. FITC
C. Alexa 488
D. Alexa 647
Select the correct combinations of the appropriate dyes that the researcher would typically utilize to observe co‐localization in an epifluorescence microscope?
PBMCs from the blood collected from a tuberculosis (TB) patient were given to four lab technicians to perform ELISPOT assay for interferon γ (IFNγ). While all steps recommended for ELISPOT were followed, the first step was performed differently by the four lab technicians, as detailed below.
A Lab technician 1 coated each well with 250.000 formaldehyde- treated cells and stimulated the cells with TB-specific antigen.
B. Lab technician 2 coated each well with 250,000 cells and did not stimulate the cells with TB-specific antigen.
C. Lab technician 3 depleted T cells from PBMCs completely, coated the wells with monocyte-enriched PBMCs, and stimulated them with TB-specific antigen.
D. Lab technician 4 coated each well with 250.000 cells and stimulated the cells with TB-specific antigens.
Which of the lab technicians assays will yield a correct ELISPOT result for interferon γ?
An anti-idiotypic antibody with fluorescent tag was used for detection of immune cells in tissue sections from a healthy individual specifically by cell surface labeling. Which of the following will have the highest chances of getting detected?
