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Amplite® Colorimetric Beta-Lactamase Activity Assay Kit
β-Lactamases are a large family of enzymes capable of hydrolyzing β-lactams. β-Lactam ring is the common element in all beta-lactam antibiotics including penicillin derivatives, cephalosporins, monobactams, and carbapenems. Through hydrolysis, β-lactamase breaks the β-lactam ring open, thus deactivates the molecule's antibacterial properties. Bacteria from clinical and non-clinical settings are becoming increasingly resistant to β-lactam antibiotics by synthesizing β-lactamase. To overcome this resistance, β-lactam antibiotics are often given with β-lactamase inhibitors such as clavulanic acid. Therefore, detection of β-lactamase activity is of central importance to assess beta-lactam antibiotics as well as to prevent antibiotics resistance. AAT Bioquest's Colorimetric Beta-Lactamase Activity Assay Kit offers a sensitive colorimetric assay for measuring β-lactamase activity in biological samples. The β-lactamase activity is detected using Nitrocefin, which changes color from yellow to red upon hydrolysis by β-lactamase. The assay can be performed using an absorbance microplate reader by measuring the OD ratio at the wavelength of 490 nm to 380 nm.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare β-Lactamase standards or test samples (50 µL)
- Add β-lactamase working solution (50 µL)
- Incubate at RT for 30 - 60 min
- Monitor absorbance increase at OD ratio of 490/380 nm
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
β-Lactamase standard stock solution (50 mU/mL)
Add 100 µL of ddH2O into the vial of β-Lactamase Standard (Component C) to make 50 mU/mL β-Lactamase standard solution.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/12551
https://www.aatbio.com/tools/serial-dilution/12551
β-Lactamase standard
Add 10 µL of 50 mU/mL β-Lactamase standard solution into 990 µL 1x PBS buffer to generate 500 µU/mL β-Lactamase standard solution (SD7). Take 500 µU/mL β-Lactamase standard solution (SD7) and perform 1:2 serial dilutions in PBS to get serial diluted β-Lactamase standard (SD6 - SD1). Note: Diluted β-Lactamase standard solution is unstable, and should be used promptly.PREPARATION OF WORKING SOLUTION
Add 50 µL Nitrocefin stock solution (Component A) into 5 mL of Assay Buffer (Component B ) and mix well to make β-Lactamase working solution. Note: This β-Lactamase working solution is enough for one 96-well plate. The β-Lactamase working solution is not stable, prepare fresh for each use.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1.Layout of β-Lactamase standards and test samples in a 96-well clear bottom microplate. SD= β-Lactamase Standards (SD1 - SD7, 7.8 to 500 µU/mL), BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well.
| BL | BL | TS | TS |
| SD1 | SD1 | ... | ... |
| SD2 | SD2 | ... | ... |
| SD3 | SD3 | ||
| SD4 | SD4 | ||
| SD5 | SD5 | ||
| SD6 | SD6 | ||
| SD7 | SD7 |
| Well | Volume | Reagent |
| SD1 - SD7 | 50 µL | Serial Dilutions (7.8 to 500 µU/mL) |
| BL | 50 µL | 1x PBS Buffer |
| TS | 50 µL | test sample |
- Prepare β-Lactamase standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of β-Lactamase working solution to each well of β-Lactamase standard, blank control, and test samples to make the total β-Lactamase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of β-Lactamase working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 30 - 60 minutes, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at OD ratio of 490/380 nm.
Citations
View all 1 citations: Citation Explorer
Circadian rhythms in the critical human pathogen Acinetobacter baumannii
Authors: Mussi, Mar{\'\i}a Alejandra and Permingeat, Valent{\'\i}n and Mora, B{\'a}rbara Perez and Migliori, Mar{\'\i}a and Arana, Natalia and Fern{\'a}ndez, Julia and Allasia, Mar{\'\i}a Bel{\'e}n and Lamberti, Melisa and Di Venanzio, Gisela and Feldman, Mario and others,
Journal: (2024)
Authors: Mussi, Mar{\'\i}a Alejandra and Permingeat, Valent{\'\i}n and Mora, B{\'a}rbara Perez and Migliori, Mar{\'\i}a and Arana, Natalia and Fern{\'a}ndez, Julia and Allasia, Mar{\'\i}a Bel{\'e}n and Lamberti, Melisa and Di Venanzio, Gisela and Feldman, Mario and others,
Journal: (2024)
References
View all 14 references: Citation Explorer
A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum beta-lactamase-producing Escherichia coli in wastewaters
Authors: Chantemesse B, Betelli L, Solanas S, Vienney F, Bollache L, Hartmann A, Rochelet M.
Journal: Water Res (2017): 375
Authors: Chantemesse B, Betelli L, Solanas S, Vienney F, Bollache L, Hartmann A, Rochelet M.
Journal: Water Res (2017): 375
Amperometric detection of extended-spectrum beta-lactamase activity: application to the characterization of resistant E. coli strains
Authors: Rochelet M, Solanas S, Betelli L, Neuwirth C, Vienney F, Hartmann A.
Journal: Analyst (2015): 3551
Authors: Rochelet M, Solanas S, Betelli L, Neuwirth C, Vienney F, Hartmann A.
Journal: Analyst (2015): 3551
Complete (1)H, (1)(5)N, and (1)(3)C resonance assignments of Bacillus cereus metallo-beta-lactamase and its complex with the inhibitor R-thiomandelic acid
Authors: Karsisiotis AI, Damblon C, Roberts GC.
Journal: Biomol NMR Assign (2014): 313
Authors: Karsisiotis AI, Damblon C, Roberts GC.
Journal: Biomol NMR Assign (2014): 313
Evaluation of phenotypic tests for the detection of AmpC beta-lactamase in clinical isolates of Escherichia coli
Authors: H, undefined and a D, P and ey A, Asthana AK, Rawat A, H and a S, Thakuria B.
Journal: Indian J Pathol Microbiol (2013): 135
Authors: H, undefined and a D, P and ey A, Asthana AK, Rawat A, H and a S, Thakuria B.
Journal: Indian J Pathol Microbiol (2013): 135
An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening
Authors: Thomas PW, Spicer T, Cammarata M, Brodbelt JS, Hodder P, Fast W.
Journal: Bioorg Med Chem (2013): 3138
Authors: Thomas PW, Spicer T, Cammarata M, Brodbelt JS, Hodder P, Fast W.
Journal: Bioorg Med Chem (2013): 3138
Page updated on November 21, 2024
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