20-Bio-A3 Biomechanics · December 2018
Nivaar worked solution (AI-drafted; not reviewed by a licensed engineer)
National Exams — December 2018 — 04-Bio-A3, Cellular and Molecular Biology and Biochemistry. Three-hour, CLOSED-BOOK exam; only an approved Casio or Sharp calculator is permitted. The paper carries six questions of equal value (20 marks each): FIVE questions constitute a complete paper and only the first five as they appear in the answer book are marked (100 marks total), with candidates urged to state any interpretive assumptions in writing. All SIX questions are worked below as a complete study resource. Question 6 is a 20-item True/False set marked +1 for a correct answer, 0 for a blank, and −1 for an incorrect answer.
Reference texts: Alberts et al., Molecular Biology of the Cell (6th ed.) — cell structure, gene regulation, DNA/RNA/protein synthesis; Nelson & Cox, Lehninger Principles of Biochemistry (7th ed.) — enzyme kinetics, Michaelis–Menten and substrate inhibition; Sambrook & Russell, Molecular Cloning: A Laboratory Manual (4th ed.) — restriction mapping, Sanger sequencing; Murphy & Weaver, Janeway's Immunobiology (9th ed.) — antibody structure and function; Murray et al., Medical Microbiology (9th ed.) — antibiotic mechanisms and susceptibility testing.
Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.
Antibodies and antibiotics differ fundamentally in molecular class, size, origin and mechanism, and every experiment below exploits one of those differences rather than trying to test for the substances directly by name.
Tests to identify an antibody. An antibody (immunoglobulin) is a large (~150 kDa for IgG), Y-shaped glycoprotein made of two heavy and two light polypeptide chains, so the diagnostic tests target its size, its protein/glycoprotein chemistry, and — most specifically — its ability to bind a particular antigen. (i) Protein content assay (e.g. Bradford or bicinchoninic acid/BCA assay): confirms the solution contains substantial protein at all, which is a necessary (but not sufficient) precondition, since essentially all classical small-molecule antibiotics would give a negative or near-negative protein assay. (ii) SDS-PAGE: run the sample under reducing and non-reducing conditions; an intact antibody runs as a single ~150 kDa band (non-reducing) that resolves into a ~50 kDa heavy-chain band and a ~25 kDa light-chain band once the inter-chain disulfides are reduced with a thiol reagent — this characteristic pattern is essentially diagnostic of an immunoglobulin and would not be produced by a small-molecule antibiotic. (iii) ELISA (enzyme-linked immunosorbent assay): coat a plate with a known antigen, add the test solution, wash, then add an enzyme-conjugated anti-immunoglobulin secondary antibody and substrate; a positive colorimetric signal demonstrates both that an antibody is present AND that it specifically binds the chosen antigen, which is the functional signature unique to antibodies. (iv) Protein A/G affinity binding: Protein A and Protein G are bacterial proteins that bind the Fc region of most IgG antibodies with high affinity and specificity; retention of the test solution's protein on a Protein A/G column (and its elution under low pH) is a rapid, specific confirmation that the protein is (or resembles) an immunoglobulin.
Tests to identify an antibiotic. An antibiotic is typically a small molecule (natural product or synthetic, generally well under 2 kDa), so the diagnostic tests target small-molecule chemical/physical properties and antimicrobial FUNCTION rather than protein structure. (i) Antimicrobial bioassay (disc-diffusion / Kirby–Bauer-style test): spot or soak a paper disc with the test solution and place it on an agar plate freshly seeded with a lawn of a known antibiotic-susceptible bacterium (e.g. Staphylococcus aureus or E. coli); after incubation, a clear zone of inhibited bacterial growth around the disc directly demonstrates antimicrobial activity, the functional hallmark of an antibiotic (an antibody solution, lacking any bactericidal/bacteriostatic small molecule, would produce no zone unless it happened to be raised against a surface antigen of that exact organism, an easily controlled confound). (ii) Mass spectrometry: determine the molecular mass of the solute; a mass in the range of roughly 150–2000 Da is consistent with essentially every classical antibiotic class (beta-lactams, aminoglycosides, macrolides, tetracyclines, etc.) and is incompatible with an intact ~150 kDa antibody. (iii) HPLC/TLC with UV or antimicrobial-class-specific colorimetric reagents: chromatographic separation and comparison of retention time/spot pattern against known antibiotic standards can identify or narrow the specific compound class present. (iv) Negative protein assay: as the converse of test (i) under "antibody" above, a negative Bradford/BCA result for protein, combined with a positive zone of inhibition in the disc-diffusion assay, is strong converging evidence for a small-molecule antibiotic rather than a protein-based antimicrobial.
Combining a positive protein/SDS-PAGE/antigen-specific-ELISA result with a negative antimicrobial bioassay identifies the solution as an antibody; conversely, a positive zone of bacterial growth inhibition combined with a small molecular mass (MS) and negative protein assay identifies it as an antibiotic. Running both panels in parallel on a single unknown sample, rather than relying on any single test, is good experimental practice because it protects against a false conclusion from any one assay's failure mode (e.g. a degraded antibody giving a weak ELISA signal, or a bacteriostatic-only antibiotic giving a smaller-than-expected inhibition zone).
