Q.22 In ELISA, which of the following enzymes are conjugated to antibodies for
detection of the analyte?
P. Alkaline phosphatase
Q. Trypsinase
R. Horseradish peroxidase
S. Amylase
(A) P and R
(B) P and Q
(C) Q and S
(D) R and S
Answer: (A) P and R
Alkaline phosphatase (P) and horseradish peroxidase (R) serve as the primary enzymes conjugated to antibodies in ELISA for analyte detection.
Option Analysis
-
P. Alkaline phosphatase: This enzyme hydrolyzes substrates like p-nitrophenyl phosphate to produce a yellow color detectable at 405 nm, making it ideal for colorimetric ELISA readouts.
-
Q. Trypsinase: No such enzyme exists in standard ELISA protocols; trypsin is a protease, not used for conjugation due to its digestive activity disrupting assays.
-
R. Horseradish peroxidase: Commonly paired with substrates like TMB for blue color development (turning yellow post-acid stop), enabling sensitive detection in various ELISA formats.
-
S. Amylase: A carbohydrate-digesting enzyme unsuitable for ELISA; it lacks chromogenic substrates stable for antibody conjugation and signal generation.
Correct Choice
Options (B), (C), and (D) include incorrect enzymes, confirming (A) as the precise match based on established immunoassay practices.
In ELISA (Enzyme-Linked Immunosorbent Assay), ELISA enzymes alkaline phosphatase horseradish peroxidase are conjugated to antibodies for precise analyte detection, enabling colorimetric, fluorescent, or chemiluminescent signals. This technique revolutionized immunoassays by replacing radioisotopes with stable enzymes, ideal for competitive exams like IIT JAM Biotechnology.
Common ELISA Enzymes
Standard enzymes include:
-
Alkaline phosphatase (AP): Generates measurable color from phosphate substrates.
-
Horseradish peroxidase (HRP): Catalyzes rapid reactions with TMB or OPD for high sensitivity.
-
Others like β-galactosidase appear rarely, but AP and HRP dominate due to substrate availability and stability.
Why Not Other Enzymes?
Proteases (e.g., trypsin, misnamed trypsinase) or amylases degrade assay components, yielding unreliable signals. Only AP and HRP offer robust conjugation and detection.
Exam-Style MCQ Breakdown
For questions like “In ELISA, which enzymes are conjugated to antibodies (P. Alkaline phosphatase, Q. Trypsinase, R. Horseradish peroxidase, S. Amylase)?”, select (A) P and R. This tests core knowledge of immunoassay reagents.
