affect the overall performance of the assay resulting in inaccurate readings.
15. The optimal antigen concentration, hamster sera dilution, and
conjugated secondary antibody dilution that give the best
signal with minimum background were determined experimentally by checkerboard analysis [25, 26].
16. The TMB substrate solution is colorless and should be stored
at 4
C. If the reagent turns blue, it may be contaminated and
should not be used for the assay. If the negative control (PBS or
no antigen) turns blue after development, the ELISA assay
should be repeated with additional washing steps.
17. The kidney is cultured immediately after it is removed from the
animal. Disperse the pulverized kidney on top of the semisolid
EMJH. Use a sterile barrier pipette tip to swirl the kidney
around to embed it in the gel.
18. Use screw-top microcentrifuge tubes to prevent tubes from
opening and samples from spilling during the homogenization
process.
19. Determine the volume (μl) of tissue to use for DNA extraction
with the following formula: x ¼ 500 μl  (25 mg)/mg of diced
tissue. For example, if 50 mg diced tissue is suspended in 500 μl
PBS, then 250 μl of homogenate will be used for DNA
extraction.
20. DNA extraction from the blood collected on EDTA must be
performed immediately after collection.
21. The standard curve is prepared with genomic DNA extracted
from the same Leptospira strain used for the challenge.
22. A genome size of 4.6 Mb was used to determine the Geq
concentration per microliter of purified DNA. A genomic
DNA mass of 50,400 pg is equivalent to 1 Â 10
7
Leptospira
genome copies [6, 24]. To prepare 1 Â 10
7 Geq/5 μl for the
standard curve, purified leptospiral genomic DNA is diluted in
distilled water to a concentration of 50,400 pg/5 μl. Tenfold
serial dilutions are prepared to obtain 1 Â 10
6 to 1 Â 10
0
Geq/5 μl.
23. In this protocol, the elution factor is 20 because we used 5 μl of
the 100 μl extracted DNA, and the volume factor is 40 because
the DNA extraction was done with 25 mg (1 g/25 mg) of
tissue. For the blood, the elution factor is 20 (5 μl of the 100 μl
DNA), and volume factor is 1 (100 μl/100 μl).
268
Karen V. Evangelista and Kristel Lourdault
15. The optimal antigen concentration, hamster sera dilution, and
conjugated secondary antibody dilution that give the best
signal with minimum background were determined experimentally by checkerboard analysis [25, 26].
16. The TMB substrate solution is colorless and should be stored
at 4
C. If the reagent turns blue, it may be contaminated and
should not be used for the assay. If the negative control (PBS or
no antigen) turns blue after development, the ELISA assay
should be repeated with additional washing steps.
17. The kidney is cultured immediately after it is removed from the
animal. Disperse the pulverized kidney on top of the semisolid
EMJH. Use a sterile barrier pipette tip to swirl the kidney
around to embed it in the gel.
18. Use screw-top microcentrifuge tubes to prevent tubes from
opening and samples from spilling during the homogenization
process.
19. Determine the volume (μl) of tissue to use for DNA extraction
with the following formula: x ¼ 500 μl  (25 mg)/mg of diced
tissue. For example, if 50 mg diced tissue is suspended in 500 μl
PBS, then 250 μl of homogenate will be used for DNA
extraction.
20. DNA extraction from the blood collected on EDTA must be
performed immediately after collection.
21. The standard curve is prepared with genomic DNA extracted
from the same Leptospira strain used for the challenge.
22. A genome size of 4.6 Mb was used to determine the Geq
concentration per microliter of purified DNA. A genomic
DNA mass of 50,400 pg is equivalent to 1 Â 10
7
Leptospira
genome copies [6, 24]. To prepare 1 Â 10
7 Geq/5 μl for the
standard curve, purified leptospiral genomic DNA is diluted in
distilled water to a concentration of 50,400 pg/5 μl. Tenfold
serial dilutions are prepared to obtain 1 Â 10
6 to 1 Â 10
0
Geq/5 μl.
23. In this protocol, the elution factor is 20 because we used 5 μl of
the 100 μl extracted DNA, and the volume factor is 40 because
the DNA extraction was done with 25 mg (1 g/25 mg) of
tissue. For the blood, the elution factor is 20 (5 μl of the 100 μl
DNA), and volume factor is 1 (100 μl/100 μl).
268
Karen V. Evangelista and Kristel Lourdault