Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay
Abstract
:1. Introduction
2. Results and Discussion
2.1. Comparison of the Sensitivity and Linear Range of Detection of the B cell Fluorospot Assay and the Traditional ELISpot Assay in Enumerating IgG and IgM ASCs
cells/well | 7B2-IgG | |||
---|---|---|---|---|
ELISpot | Fluorospot | |||
ASC/wella | spot:cell ratio | ASC/well a | spot:cell ratio | |
0 | 0 | n/a | 0 | n/a |
100 | 52 ± 9 | 0.52 | 50 ± 11 | 0.50 |
500 | 232 ± 19 | 0.46 | 222 ± 18 | 0.44 |
1,000 | 416 ± 18 | 0.42 | 417 ± 12 | 0.42 |
5,000 | 1,669 ± 260 | 0.33 | 1,721 ± 64 | 0.34 |
Linearity of the range of detection | ||||
r | 0.99<0.0001 | 0.998<0.0001 | ||
p |
cells/well | 2B9-IgM | |||
---|---|---|---|---|
ELISpot | Fluorospot | |||
ASC/well a | spot:cell ratio | ASC/well a | spot:cell ratio | |
0 | 0 | n/a | 0 | n/a |
100 | 9 ± 1 | 0.09 | 17 ± 0 | 0.17 |
500 | 52 ± 2 | 0.10 | 67 ± 1 | 0.13 |
1,000 | 95 ± 4 | 0.10 | 138 ± 8 | 0.14 |
5,000 | 453 ± 11 | 0.09 | 518 ± 22 | 0.10 |
Linearity of the range of detection | ||||
r | 0.999<0.0001 | 0.997<0.0001 | ||
p |
2.2. Multiplex Detection of IgG and IgM Antibody-Secreting Cells
Condition | cells/well | IgG ASCs | IgM ASCs | |||||||
---|---|---|---|---|---|---|---|---|---|---|
IgG-7B2 | IgM-2B9 | Well 1 | Well 2 | Average | Well 1 | Well 2 | Average | |||
Experiment 1 | ||||||||||
1:0 | 1,000 | 0 | 955 | 987 | 971 | 0 | 0 | 0 | ||
1:0.5 | 1,000 | 500 | 915 | 906 | 911 | 43 | 47 | 45 | ||
1:1 | 1,000 | 1,000 | 827 | 940 | 884 | 79 | 77 | 78 | ||
0.5:1 | 500 | 1,000 | 465 | 527 | 496 | 69 | 75 | 72 | ||
0:1 | 0 | 1,000 | 0 | 0 | 0 | 76 | 67 | 72 | ||
Experiment 2 | ||||||||||
1:0 | 500 | 0 | 360 | 325 | 343 | 0 | 0 | 0 | ||
1:0.5 | 500 | 1,000 | 343 | 334 | 339 | 94 | 92 | 93 | ||
1:1 | 500 | 2,000 | 304 | 365 | 335 | 175 | 145 | 160 | ||
0.5:1 | 250 | 2,000 | 161 | 140 | 151 | 178 | 198 | 188 | ||
0:1 | 0 | 2,000 | 0 | 0 | 0 | 195 | 183 | 189 |
2.3. Multiplex Detection of HIV-1 gp41 Env-Specific ASCs
2.4. Discussion
3. Experimental Section
3.1. Cell Lines
3.2. Conjugation of Anti-Human IgG and IgM to Quantum-Dot Nanocrystals
3.3. B Cell Fluorospot Assay
3.4. Acquisition of Fluorescent Images
3.5. Traditional ELISpot Assay
4. Conclusions
Acknowledgments
Conflict of Interest
References
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Bonsignori, M.; Moody, M.A. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay. Cells 2012, 1, 15-26. https://doi.org/10.3390/cells1010015
Bonsignori M, Moody MA. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay. Cells. 2012; 1(1):15-26. https://doi.org/10.3390/cells1010015
Chicago/Turabian StyleBonsignori, Mattia, and M. Anthony Moody. 2012. "Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay" Cells 1, no. 1: 15-26. https://doi.org/10.3390/cells1010015