routinely use 0.2 as it is sufficiently higher than the absorbance
reading we normally obtain in background wells with no or
naı ¨ve sample (i.e., 0.05). It is also >fourfold lower than the
peak values we obtain when signal levels may be saturated. If a
particular sample does not reach the target OD, we suggest
assigning the value of the lowest tested dilution for that particular sample for analysis/presentation purposes.
16. Titers should not be directly compared between different
assays (different coating antigens, antibody isotypes, subtypes,
etc.). The titers within a particular assay depend on the concentrations/activities of various assay reagents, such as coating
protein and detection antibodies. This assay will allow you to
rank antibody titers within a particular assay, but will not
provide fixed concentrations or amounts of antibody. In
order to do this, a standard curve is required using samples
with a known antibody concentration. Another possible advantage of including a standard curve is the potential to assay more
samples per plate. As the aim of the ELISA shifts from calculating a target OD to finding a value that falls within the range of a
standard curve, a smaller number of sample dilutions can be
tested per sample.
17. A “hook effect” may be observed at the lower end of the
dilution range where OD values appear lower than in more
diluted samples before they start decreasing as would be
expected with subsequent dilutions. This may be due to inhibitory effects of the serum matrix when added at high concentrations or when all available binding sites have been saturated.
Acknowledgments
The authors would like to acknowledge Renu Dudani and Blair
Harrison for their technical assistance with the development of this
assay.
References
1. Plotkin SA (2008) Vaccines: correlates of
vaccine-induced immunity. Clin Infect Dis
47:401–409
2. Akache B, Stark FC, Iqbal U, Chen W, Jia Y,
Krishnan L, McCluskie MJ (2018) Safety and
biodistribution of sulfated archaeal glycolipid
archaeosomes as vaccine adjuvants. Hum Vaccin Immunother 14(7):1746–1759
3. Akache B, Stark FC, Jia Y, Deschatelets L,
Dudani R, Harrison BA, Agbayani G,
Williams D, Jamshidi MP, Krishnan L et al
(2018)
Sulfated
archaeol
glycolipids:
comparison with other immunological adjuvants in mice. PLoS One 13:e0208067
4. Jia Y, Akache B, Deschatelets L, Qian H,
Dudani R, Harrison BA, Stark FC,
Chandan V, Jamshidi MP, Krishnan L et al
(2019) A comparison of the immune responses
induced by antigens in three different
archaeosome-based vaccine formulations. Int
J Pharm 561:187–196
5. Pavliakova D, Giardina PC, Moghazeh S,
Sebastian S, Koster M, Pavliak V, McKeen A,
French R, Jansen KU, Pride M (2018)
546
Bassel Akache et al.
reading we normally obtain in background wells with no or
naı ¨ve sample (i.e., 0.05). It is also >fourfold lower than the
peak values we obtain when signal levels may be saturated. If a
particular sample does not reach the target OD, we suggest
assigning the value of the lowest tested dilution for that particular sample for analysis/presentation purposes.
16. Titers should not be directly compared between different
assays (different coating antigens, antibody isotypes, subtypes,
etc.). The titers within a particular assay depend on the concentrations/activities of various assay reagents, such as coating
protein and detection antibodies. This assay will allow you to
rank antibody titers within a particular assay, but will not
provide fixed concentrations or amounts of antibody. In
order to do this, a standard curve is required using samples
with a known antibody concentration. Another possible advantage of including a standard curve is the potential to assay more
samples per plate. As the aim of the ELISA shifts from calculating a target OD to finding a value that falls within the range of a
standard curve, a smaller number of sample dilutions can be
tested per sample.
17. A “hook effect” may be observed at the lower end of the
dilution range where OD values appear lower than in more
diluted samples before they start decreasing as would be
expected with subsequent dilutions. This may be due to inhibitory effects of the serum matrix when added at high concentrations or when all available binding sites have been saturated.
Acknowledgments
The authors would like to acknowledge Renu Dudani and Blair
Harrison for their technical assistance with the development of this
assay.
References
1. Plotkin SA (2008) Vaccines: correlates of
vaccine-induced immunity. Clin Infect Dis
47:401–409
2. Akache B, Stark FC, Iqbal U, Chen W, Jia Y,
Krishnan L, McCluskie MJ (2018) Safety and
biodistribution of sulfated archaeal glycolipid
archaeosomes as vaccine adjuvants. Hum Vaccin Immunother 14(7):1746–1759
3. Akache B, Stark FC, Jia Y, Deschatelets L,
Dudani R, Harrison BA, Agbayani G,
Williams D, Jamshidi MP, Krishnan L et al
(2018)
Sulfated
archaeol
glycolipids:
comparison with other immunological adjuvants in mice. PLoS One 13:e0208067
4. Jia Y, Akache B, Deschatelets L, Qian H,
Dudani R, Harrison BA, Stark FC,
Chandan V, Jamshidi MP, Krishnan L et al
(2019) A comparison of the immune responses
induced by antigens in three different
archaeosome-based vaccine formulations. Int
J Pharm 561:187–196
5. Pavliakova D, Giardina PC, Moghazeh S,
Sebastian S, Koster M, Pavliak V, McKeen A,
French R, Jansen KU, Pride M (2018)
546
Bassel Akache et al.