To minimize the risk of microbial growth, the solution may be
autoclaved or stored at 2–8
C. As salt precipitates may form
with the latter option, it is important to warm up the 10Â
solution to ensure complete dissolution of the buffer components prior to dilution with water to obtain the working 1Â
Wash buffer solution.
3. Exercise great caution when diluting the sulfuric acid for the
stop solution. Be sure to work in a chemical hood with proper
ventilation and follow all appropriate safety precautions. Also,
dispense acid slowly into the bottle already containing the
entire volume of water so as not to generate too much heat/
energy. Allow solution to cool down before firmly tightening
the lid. The stop solution should be stored in an appropriate
acid cabinet.
4. Enzyme immunoassay plates are used as they are designed to
maintain assay consistency and improve binding of the coating
antigen to the plate surface. As this is a colorimetric assay,
transparent plates are used to allow measurement of the solution OD by the spectrophotometer.
5. If preferred, plasma samples may also be used to measure
antigen-specific antibody titers. While a strong correlation in
titers against some antigens has been demonstrated with
plasma and serum collected simultaneously [10], it would be
prudent to select one sample type in a particular experiment if
possible.
6. The ELISA will be conducted in a 96-well format, where
multichannel pipettes will be employed to dilute the serum.
Therefore, when dealing with a large number of samples, it is
more efficient to collect and store samples in a format compatible with a multichannel pipette (e.g., 8 or 12- cluster strips of
1.2 mL). The samples can be arranged to match your final
layout (e.g., leave columns 11 and 12 empty to have room in
the plate for controls). If transferring the samples with a multichannel pipette, confirm that all tips contain equal volume
before loading onto the dilution plate.
7. Coating concentration will need to be optimized to your particular antigen type/source. A trial run can be conducted with
various concentrations of antigen. Ideally, you should select a
concentration where the signal is close to saturation with the
positive samples (i.e., increasing coating concentration will not
boost your signal significantly) and where the negative control
results in a low background signal (OD 450 < 0.1).
8. Once the washing step is complete, tap plates while upside
down onto paper towel to remove any residual wash solution
remaining in the wells prior to addition of the subsequent
solution (Blocking Buffer, samples, etc.).
544
Bassel Akache et al.
autoclaved or stored at 2–8
C. As salt precipitates may form
with the latter option, it is important to warm up the 10Â
solution to ensure complete dissolution of the buffer components prior to dilution with water to obtain the working 1Â
Wash buffer solution.
3. Exercise great caution when diluting the sulfuric acid for the
stop solution. Be sure to work in a chemical hood with proper
ventilation and follow all appropriate safety precautions. Also,
dispense acid slowly into the bottle already containing the
entire volume of water so as not to generate too much heat/
energy. Allow solution to cool down before firmly tightening
the lid. The stop solution should be stored in an appropriate
acid cabinet.
4. Enzyme immunoassay plates are used as they are designed to
maintain assay consistency and improve binding of the coating
antigen to the plate surface. As this is a colorimetric assay,
transparent plates are used to allow measurement of the solution OD by the spectrophotometer.
5. If preferred, plasma samples may also be used to measure
antigen-specific antibody titers. While a strong correlation in
titers against some antigens has been demonstrated with
plasma and serum collected simultaneously [10], it would be
prudent to select one sample type in a particular experiment if
possible.
6. The ELISA will be conducted in a 96-well format, where
multichannel pipettes will be employed to dilute the serum.
Therefore, when dealing with a large number of samples, it is
more efficient to collect and store samples in a format compatible with a multichannel pipette (e.g., 8 or 12- cluster strips of
1.2 mL). The samples can be arranged to match your final
layout (e.g., leave columns 11 and 12 empty to have room in
the plate for controls). If transferring the samples with a multichannel pipette, confirm that all tips contain equal volume
before loading onto the dilution plate.
7. Coating concentration will need to be optimized to your particular antigen type/source. A trial run can be conducted with
various concentrations of antigen. Ideally, you should select a
concentration where the signal is close to saturation with the
positive samples (i.e., increasing coating concentration will not
boost your signal significantly) and where the negative control
results in a low background signal (OD 450 < 0.1).
8. Once the washing step is complete, tap plates while upside
down onto paper towel to remove any residual wash solution
remaining in the wells prior to addition of the subsequent
solution (Blocking Buffer, samples, etc.).
544
Bassel Akache et al.