Bioassays are assays or biological techniques to measure strength, potency, concentration or efficacy of any substance by its effect on biological substance like tissues, cells, animals or enzymes etc

1. BIOASSAY FOR PROTEINS,
VITAMINS AND ANTIBIOTICS
Sourabh Santosh Gurav
M.Tech Bioprocess Technology
20BPT218
Analytical Techniques in Bioprocessing
Bioassays for Protein, Vitamins & Antibiotics 1

2. BIOASSAY : INTRODUCTION AND
PRINCIPLE
‣Analytical method to determine concentration, potency, purity or
strength of a substance by its effect on living cells, tissues or
biologically derived molecules (Receptors, enzymes, Antibodies etc)
‣Qualitative or Quantitative
‣Direct or Indirect
‣Intensity of stimulus is varied by doses and depending on this intensity
of stimulus, a change/response will be followed by a subject
Bioassays for Protein, Vitamins & Antibiotics 2

3. MEASUREMENT OF RESPONSE OF THE
BIOASSAY
 Turbidomentry (16 to 24 hours incubation for microbial growth)
 Gravimetry (Fungal growth measurement)
 Direct cell count in haemocytometer.
 Determination of pH change (after 18 hrs of lactic acid bacteria)
 Measuring diffusion zone (Death)
 Fluorescent or radioactive signals from coupled reactions (ELISA,
BiFC, RIA)
Bioassays for Protein, Vitamins & Antibiotics 3

4. BIOASSAY FOR PROTEINS
ENZYME-LINKED IMMUNOSORBENT ASSAY
‣ ELISA detects presence and concn of antigens in biological samples
‣ Antigen is immobilized to a solid surface (directly, or indirectly via
capture antibody)
‣ Antigen complexed to a detection antibody conjugated with a
molecule amenable for detection - an enzyme or a fluorophore.
‣ Requirement : multi-well plate (96- or 384-wells)
Bioassays for Protein, Vitamins & Antibiotics 4
QUANTITATIVE QUALITATIVE
• comparing them to a standard
curve.
• mean absorbance (y axis) against
the protein concentration (x axis)
• Data is a yes or a no answer
• Confirms or denies whether the
presence of a particular antigen

5. Bioassays for Protein, Vitamins & Antibiotics 5
Direct ELISA Indirect ELISA Sandwich ELISA
PRINCIPLE OF ELISA ABSORBANCE-CONCENTRATION STANDARD CURVE

6. BIOASSAY FOR PROTEINS
Radioimmunoassay for Ag, Abs and proteins
‣ RIA detects Ag or substrates in samples using
radio-labelled molecules.
‣ 125-I attached to Tyrosine
‣ Amount of radiation is detected in a gamma
counter
‣ Radiolabeled antigen is mixed with a known
amount of immobilized Ab for that antigen
‣ sample from a patient containing an unknown
quantity of that same antigen is added
‣ cold" Ag is increased, more of it binds to Ab,
displacing radiolabeled variant
Bioassays for Protein, Vitamins & Antibiotics 6
STANDARD CURVE AND BASELINE VALUES FROM THE
17B-ESTRADIOL RADIOIMMUNOASSAY

7. BIOASSAY FOR PROTEINS
Bimolecular fluorescence complementation in Cell
‣ Bimolecular fluorescence complementation - BiFC
‣ Validate protein interactions
‣ Principle : association of fluorescent protein
fragments attached to components of the
same macromolecular complex
‣ Interaction of these proteins allows reporter protein
to reform in its native three-dimensional structure
and emit its fluorescent signal
‣ Detection and localization within the cell using
an inverted fluorescence microscope
Bioassays for Protein, Vitamins & Antibiotics 7
NON-FLUORESCENT FRAGMENTS (YN AND YC)
AND
PUTATIVE INTERACTION PARTNERS (A AND B)

8. BIOASSAY FOR PROTEINS
Bimolecular fluorescence complementation in cell
‣ APPLICATIONS
1. Record ribosome biogenesis
events in E.coli
2. Visualization of multiple protein
complexes to be visualized
simultaneously in the same cell
3. Study of RNA-binding protein
interactions
4. Strength of the protein
interaction can be quantitatively
determined by changes in
fluorescent signal strength
Bioassays for Protein, Vitamins & Antibiotics 8
NEURON FLUORESCENCE LIGHT
MICROGRAPH
40X-600X TRINOCULAR INVERTED
FLUORESCENCE MICROSCOPE WITH
PHASE-CONTRAST

9. BIOASSAY FOR ANTIBIOTICS
KIRBY-BAUER DISK DIFFUSION METHOD
‣ Bacterium is swabbed on the agar and the
antibiotic discs are placed on top
‣ Filter paper disks impregnated with
standardized concentration of antimicrobial
agent placed on the surface
‣ Size of the zone of inhibition around the disk
is measured
‣ Results are noted as Resistant, Intermediate &
Susceptible
Bioassays for Protein, Vitamins & Antibiotics 9
KIRBY-BAUER TEST RESULTS
https://bio.libretexts.org/

10. BIOASSAY FOR ANTIBIOTICS
MIC DETERMINATION BY MICROTITRE BROTH DILUTION
Bioassays for Protein, Vitamins & Antibiotics 10
‣ Inoculum size for standard MIC is 104 to
105 CFU/ml
‣ Lowest concentration of drug that fails to show
any growth on subculture plate is considered as
the MBC of the antibiotic for strain.
‣ 96-welled (12 x 8) micro-titre plate
‣ Aliquot of 0.5% of 2,3,5-triphenyl tetrazolium
chloride (TTC) is added , giving pink coloration
due to bacterial growth
‣ Instrument : Microplate Reader ( detection
mode Absorbance)
DILUTION OF DRUG IN THE BROTH

11. Bioassays for Protein, Vitamins & Antibiotics 11
ANTIBIOTIC SUSCEPTIBILITY TESTING
by ETEST® Ceftolozane/Tazobactam (C/T 256)
• CELL-BASED VIABILITY ASSAYS USING RESAZURIN
Amount of resorufin can be monitored by measuring fluorescence or absorbance
Resazurin (blue and non-fluorescent) is reduced by dehydrogenase enzymes to
form the red fluorescent dye resorufin

12. BIOASSAY FOR ANTIBIOTICS
IMPEDANCE-BASED FAST ANTIMICROBIAL SUSCEPTIBILITY TEST
• Cells suspended in an electrolyte flow along the channel one-by-one
through two pairs of electrodes.
• Technique measures the electrical properties of single particles as they
flow between microelectrodes within a microfluidic chip
• Electrodes are driven by an AC signal of several frequencies and when
a cell flows along the channel it perturbs the AC current
Bioassays for Protein, Vitamins & Antibiotics
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13. BIOASSAY FOR VITAMINS
MICROBIOLOGICAL ASSAY FOR VITAMIN B3 & B9
‣ Growth of microorganisms is
proportional to requirement of
vitamins
‣ Microbial solutions are treated with
samples
‣ Growth quantified by turbidimetry at
A560-200 nm
‣ Lactobacillus Plantarum – Niacin
(B3)
‣ Lactobacillus rhamnosus – Folate
(B9)
‣ ELISA reader is required for
evaluation of the folic acid content
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Folic Acid Microtiter Plate Assay
Kit by Eagle Biosciences

14. Bioassays for Protein, Vitamins & Antibiotics 14
GROWTH RATE MEASURED AS A595nm IN RESPONSE
TO VIT. B12
CORRELATION BETWEEN ZONE DIAMETER AND
MINIMUM INHIBITORY CONCENTRATION
(spearman's rank correlation coefficient = −0.549)
INDIAN JOURNAL OF MEDICAL MICROBIOLOGY
VITAMIN B12 ANTIOBIOTIC - AZITHROMYCIN

15. BIOASSAY FOR VITAMINS
RAT ASSAY METHOD FOR VIT. B2
• Growth response to graded doses of riboflavin in male albino rats
• Growth increments due to vitamin are linear between 3 & 9 μgm/day
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16. REFERENCES
‣ Kerppola T. K. (2008). Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein
interactions in living cells. Annual review of biophysics, 37, 465–487.
https://doi.org/10.1146/annurev.biophys.37.032807.125842
‣ Boutonnier, A., Dassy, B., Duménil, R., Guénolé, A., Ratsitorahina, M., Migliani, R., & Fournier, J. M.
(2003). A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio
cholerae O1 and Vibrio cholerae O139. Journal of microbiological methods, 55(3), 745–753.
https://doi.org/10.1016/j.mimet.2003.08.010
‣ Bayot, M. L., & Bragg, B. N. (2020). Antimicrobial Susceptibility Testing. In StatPearls. StatPearls
Publishing.
‣ Vega-Avila, E., & Pugsley, M. K. (2011). An overview of colorimetric assay methods used to assess survival
or proliferation of mammalian cells. Proceedings of the Western Pharmacology Society, 54, 10–14.
‣ Determination of Vitamin B2 (Riboflavin): Comparison of Bioassay, microbiological, and Fluorometric
Methods, A Emmett, O Bird, R Brown, Gail Peacock, and J Vandenbelt; Industrial & Engineering Chemistry
Analytical Edition 1941 13 (4), 219-221, DOI: 10.1021/i560092a004
‣ Cheung, K. C., Di Berardino, M., Schade-Kampmann, G., Hebeisen, M., Pierzchalski, A., Bocsi, J., Mittag,
A., & Tárnok, A. (2010). Microfluidic impedance-based flow cytometry. Cytometry. Part A : the journal of
the International Society for Analytical Cytology, 77(7), 648–666. https://doi.org/10.1002/cyto.a.20910
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