Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Reagents
2.2. Serum Samples
2.3. Luminescent-Based SBA (L-SBA) in 96- and 384-Well Plates
2.4. Calculations
3. Results
3.1. Moving from 96- to 384-Well Plates
3.2. Comparison between L-SBA in 96- and 384-Well Plates
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species and Serovar | Strain | Characteristics | Reference(s) |
---|---|---|---|
Shigella flexneri serotype 1b | 140 | Clinical isolate | Public Health England (PHE) |
Shigella flexneri serotype 2a | 142 | Clinical isolate | Public Health England (PHE) |
Shigella flexneri serotype 3a | 144 | Clinical isolate | Public Health England (PHE) |
Shigella sonnei | 71 | S. sonnei 53G ΔvirG::cat | [26] |
Citrobacter freundii | NVGH328 | Clinical isolate from Novartis Master Culture | [27] |
Salmonella enterica serovar Typhimurium | D23580 | Clinical isolate from blood culture, Malawi | [28,29] |
Salmonella enterica serovar Enteritidis | CMCC4314 | (corresponding to ATCC4931) obtained from the Novartis Master Culture Collection (NMCC) | [30] |
Salmonella Paratyphi A | NVGH308 | Invasive isolates, Nepal | [31] |
L-SBA Titers (IC50) | L-SBA Titers (IC50) | |||||||
---|---|---|---|---|---|---|---|---|
96-Wells-Plate SBA | 384-Wells-Plate SBA | 96-Wells-Plate SBA | 384-Wells-Plate SBA | |||||
S. flexneri1b Strain | Mouse Standard Antiserum | Rep. 1 | 8126 | 8490 | Rabbit Standard Antiserum | Rep. 1 | 24,732 | 29,776 |
Rep. 2 | 7738 | 12,255 | Rep. 2 | 25,699 | 31,448 | |||
Rep. 3 | 8257 | 10,670 | Rep. 3 | 33,031 | 26,493 | |||
Rep. 4 | 9092 | 8694 | Rep. 4 | 46,997 | 28,192 | |||
Rep. 5 | 8697 | 9869 | Rep. 5 | 48,571 | 24,008 | |||
Rep. 6 | 10,322 | 8784 | Rep. 6 | 40,635 | 25,847 | |||
Rep. 7 | 8965 | 13,089 | Rep. 7 | 23,901 | 45,903 | |||
Rep. 8 | 8533 | 12,554 | Rep. 8 | 28,454 | ||||
GeoMean | 8687 | 10,406 | GeoMean | 33,418 | 29,456 | |||
SE | 260 | 621 | SE | 3481 | 2258 | |||
SE% | 3 | 6 | SE% | 10 | 8 | |||
S. flexneri2a strain | Mouse standard antiserum | Rep. 1 | 8119 | 8905 | Rabbit Standard antiserum | Rep. 1 | 8770 | 8380 |
Rep. 2 | 8473 | 9682 | Rep. 2 | 11,246 | 9054 | |||
Rep. 3 | 8477 | 22,253 | Rep. 3 | 12,632 | 18,019 | |||
Rep. 4 | 8450 | 15,172 | Rep. 4 | 19,843 | 14,242 | |||
Rep. 5 | 8657 | 9233 | Rep. 5 | 10,420 | 8821 | |||
Rep. 6 | 8741 | 11,761 | Rep. 6 | 10,428 | 16,238 | |||
Rep. 7 | 8591 | 11,323 | Rep. 7 | 9104 | 10,757 | |||
Rep. 8 | 8376 | 12,974 | Rep. 8 | 7926 | 11,323 | |||
GeoMean | 8484 | 12,116 | GeoMean | 10,863 | 11,652 | |||
SE | 63 | 1456 | SE | 1243 | 1206 | |||
SE% | 1 | 12 | SE% | 11 | 10 | |||
S. flexneri3a Strain | mouse Standard Antiserum | Rep. 1 | 46,819 | 24,862 | Rabbit standard antiserum | Rep. 1 | 8811 | 12,624 |
Rep. 2 | 25,205 | 70,660 | Rep. 2 | 11,374 | 21,178 | |||
Rep. 3 | 43,233 | 56,883 | Rep. 3 | 11,343 | 23,076 | |||
Rep. 4 | 27,388 | 40,699 | Rep. 4 | 16,600 | 17,792 | |||
Rep. 5 | 39,309 | 25,032 | Rep. 5 | 16,928 | 7450 | |||
Rep. 6 | 34,009 | 24,001 | Rep. 6 | 9086 | 8619 | |||
Rep. 7 | 32,125 | 24,675 | Rep. 7 | 8417 | 8149 | |||
Rep. 8 | 26,146 | 23,684 | Rep. 8 | 8141 | 8387 | |||
GeoMean | 33,454 | 33,063 | GeoMean | 10,897 | 12,171 | |||
SE | 2691 | 6030 | SE | 1180 | 2110 | |||
SE% | 8 | 18 | SE% | 11 | 17 | |||
S. sonneiStrain | Mouse Standard Antiserum | Rep. 1 | 1658 | 2517 | Rabbit standard antiserum | Rep. 1 | 6630 | 17,512 |
Rep. 2 | 1150 | 2290 | Rep. 2 | 8097 | 9570 | |||
Rep. 3 | 2357 | 2081 | Rep. 3 | 8673 | 12,372 | |||
Rep. 4 | 1529 | 2460 | Rep. 4 | 9433 | 18,587 | |||
Rep. 5 | 1139 | 2489 | Rep. 5 | 16,669 | 18,851 | |||
Rep. 6 | 1121 | 1068 | Rep. 6 | 15,471 | 9528 | |||
Rep. 7 | 2014 | 2160 | Rep. 7 | 8789 | 12,956 | |||
Rep. 8 | 2258 | 1054 | Rep. 8 | 8342 | 14,005 | |||
GeoMean | 1585 | 1912 | GeoMean | 9770 | 13,721 | |||
SE | 168 | 201 | SE | 1219 | 1250 | |||
SE% | 11 | 11 | SE% | 12 | 9 | |||
S.Typhimurium strain | Mouse standard antiserum | Rep. 1 | 28,133 | 16,038 | Anti-rabbit standard serum | Rep. 1 | 61,185 | 25,018 |
Rep. 2 | 21,515 | 23,282 | Rep. 2 | 40,788 | 24,404 | |||
Rep. 3 | 21,259 | 9986 | Rep. 3 | 27,883 | 25,895 | |||
Rep. 4 | 22,669 | 14,926 | Rep. 4 | 28,813 | 23,882 | |||
Rep. 5 | 25,324 | 18,654 | Rep. 5 | 44,251 | 23,305 | |||
Rep. 6 | 23,509 | 9197 | Rep. 6 | 27,725 | 21,824 | |||
Rep. 7 | 23,182 | 9208 | Rep. 7 | 26,312 | 16,567 | |||
Rep. 8 | 8749 | Rep. 8 | 63,920 | 23,675 | ||||
GeoMean | 23,557 | 12,905 | GeoMean | 37,765 | 22,888 | |||
SE | 783 | 1777 | SE | 5077 | 956 | |||
SE% | 3 | 14 | SE% | 13 | 4 | |||
S.Enteritidis strain | Mouse standard antiserum | Rep. 1 | 7984 | 18,043 | Rabbit standard antiserum | Rep. 1 | 14,317 | 8527 |
Rep. 2 | 8327 | 11,270 | Rep. 2 | 8448 | 9505 | |||
Rep. 3 | 6037 | 16,781 | Rep. 3 | 8931 | 8681 | |||
Rep. 4 | 7377 | 13,950 | Rep. 4 | 22,548 | 27,381 | |||
Rep. 5 | 5105 | 15,416 | Rep. 5 | 25,966 | 25,482 | |||
Rep. 6 | 4482 | 8436 | Rep. 6 | 22,255 | 9927 | |||
Rep. 7 | 7463 | 8867 | Rep. 7 | 9179 | 9091 | |||
Rep. 8 | 7867 | 8457 | Rep. 8 | 8701 | 8442 | |||
GeoMean | 6683 | 12,116 | GeoMean | 13,532 | 11,793 | |||
SE | 475 | 1293 | SE | 2448 | 2675 | |||
SE% | 7 | 11 | SE% | 18 | 23 | |||
S.Paratyphi strain | Mouse standard antiserum | Rep. 1 | 6012 | 6008 | Rabbit standard antiserum | Rep. 1 | 1400 | 941 |
Rep. 2 | 2962 | 4244 | Rep. 2 | 851 | 1296 | |||
Rep. 3 | 6090 | 6353 | Rep. 3 | 865 | 974 | |||
Rep. 4 | 7579 | 6832 | Rep. 4 | 968 | 998 | |||
Rep. 5 | 9471 | 5474 | Rep. 5 | 1748 | 1361 | |||
Rep. 6 | 8381 | 5657 | Rep. 6 | 1065 | 775 | |||
Rep. 7 | 4740 | 4694 | Rep. 7 | 924 | 1201 | |||
Rep. 8 | 6259 | 7945 | Rep. 8 | 912 | 1026 | |||
GeoMean | 6107 | 5798 | GeoMean | 1058 | 1056 | |||
SE | 680 | 389 | SE | 105 | 65 | |||
SE% | 11 | 7 | SE% | 10 | 6 | |||
C. freundiistrain | Mouse standard antiserum | Rep. 1 | 11,600 | 12,664 | Rabbit standard antiserum | Rep. 1 | 1216 | 959 |
Rep. 2 | 9214 | 10,450 | Rep. 2 | 1579 | 1317 | |||
Rep. 3 | 14,273 | 11,422 | Rep. 3 | 1693 | 1503 | |||
Rep. 4 | 9719 | 12,792 | Rep. 4 | 1665 | 1490 | |||
Rep. 5 | 10,345 | 14,563 | Rep. 5 | 1634 | 1588 | |||
Rep. 6 | 8914 | 12,121 | Rep. 6 | 1629 | 1222 | |||
Rep. 7 | 7585 | 11,946 | Rep. 7 | 1539 | 1300 | |||
Rep. 8 | 12,104 | Rep. 8 | 1195 | |||||
GeoMean | 10,052 | 12,209 | GeoMean | 1557 | 1307 | |||
SE | 710 | 393 | SE | 53 | 67 | |||
SE% | 7 | 3 | SE% | 3 | 5 |
Traditional CFU-Based SBA | 96-Wells-Plate L-SBA | 384-Wells-Plate L-SBA | |
---|---|---|---|
Final Volume Reaction | 100 µL/well | 100 µL/well | 50 µL/well |
Assay Time | 1.5 working day | 6 h | 6 h |
Plate Reading | 2–3 h/SBA plate | 2 min/SBA plate | 5 min/SBA plate |
Reproducibility | Lower operator independence (for manual CFU counting) than 96- and 384-wells L-SBA | High operator independence | Higher operator independence than 96-well L-SBA |
Throughput | Plates/day: 2 | Plates/day: 8 | Plates/day: 4 (equivalent to sixteen 96-wells plates) |
1 operator/1.5 day: 22 individual sera in single | 1 operator/day: 88 individual sera in single | 1 operator/day: 188 individual sera in single | |
Reagent Costs | 4 EUR/serum | 12 EUR/serum | 8 EUR/serum |
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Aruta, M.G.; Carducci, M.; Micoli, F.; Necchi, F.; Rossi, O. Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA). BioTech 2021, 10, 19. https://doi.org/10.3390/biotech10030019
Aruta MG, Carducci M, Micoli F, Necchi F, Rossi O. Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA). BioTech. 2021; 10(3):19. https://doi.org/10.3390/biotech10030019
Chicago/Turabian StyleAruta, Maria Grazia, Martina Carducci, Francesca Micoli, Francesca Necchi, and Omar Rossi. 2021. "Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)" BioTech 10, no. 3: 19. https://doi.org/10.3390/biotech10030019
APA StyleAruta, M. G., Carducci, M., Micoli, F., Necchi, F., & Rossi, O. (2021). Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA). BioTech, 10(3), 19. https://doi.org/10.3390/biotech10030019