2.5. Quantitati6e analysis of ABA
Quantitative analysis of ABA was performed by
ELISA with rabbit polyclonal antibody raised against mouse IGg (RAMIG) and anti-ABA
mouse, monoclonal antibody (MAB). ABA labelled
alkaline phosphatase and Sigma 104 phosphatase
substrate (p-Nitrophenyl phosphate,
disodium, hexahydrate) were used as a tracer and
substrate, respectively. The assay was run as described
by Weiler [18] and Mertens et al. [19] using
96 wells ELISA plates (Maxisorb, NUNC) and
ODs were read by an ELISA reader Dynatech
MR 5000 under Revelation 2.0 control. For each
plate a standard curve was obtained. According to
Mertens et al. [19] the anti-ABA MAB shows
100% reactivity with 2-cis-(s)-ABA and very low
B0.1% cross reactivity with compounds structurally
similar to ABA. Professor E.W. Weiler
(Ruhr University, Bochum, Germany) generously
provided the antibody and tracer as a gift. In
order to check whether samples contained immunoreactive
compounds other than ABA, several
dilutions of the standard curve were spiked with
three dilutions of samples with the highest and
lowest concentration of ABA. The spiked standard
curves were parallel to the original one in its linear
range (data not presented). In most cases approximately
1 g of fresh weight (FW) of tissue (in at
least three replications) was used for extraction.
For each sample ABA was assayed in at least
three dilutions each repeated three times. Data
were analysed by ANOVA 1 and the differences
were estimated using Student’s t-test.
2.5. Quantitati6e analysis of ABA
Quantitative analysis of ABA was performed by
ELISA with rabbit polyclonal antibody raised against mouse IGg (RAMIG) and anti-ABA
mouse, monoclonal antibody (MAB). ABA labelled
alkaline phosphatase and Sigma 104 phosphatase
substrate (p-Nitrophenyl phosphate,
disodium, hexahydrate) were used as a tracer and
substrate, respectively. The assay was run as described
by Weiler [18] and Mertens et al. [19] using
96 wells ELISA plates (Maxisorb, NUNC) and
ODs were read by an ELISA reader Dynatech
MR 5000 under Revelation 2.0 control. For each
plate a standard curve was obtained. According to
Mertens et al. [19] the anti-ABA MAB shows
100% reactivity with 2-cis-(s)-ABA and very low
B0.1% cross reactivity with compounds structurally
similar to ABA. Professor E.W. Weiler
(Ruhr University, Bochum, Germany) generously
provided the antibody and tracer as a gift. In
order to check whether samples contained immunoreactive
compounds other than ABA, several
dilutions of the standard curve were spiked with
three dilutions of samples with the highest and
lowest concentration of ABA. The spiked standard
curves were parallel to the original one in its linear
range (data not presented). In most cases approximately
1 g of fresh weight (FW) of tissue (in at
least three replications) was used for extraction.
For each sample ABA was assayed in at least
three dilutions each repeated three times. Data
were analysed by ANOVA 1 and the differences
were estimated using Student’s t-test.
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