Enzyme-Linked Immunosorbent Assay (ELISA) was used to quantify the Cry1Ab concentration in the same leaf samples that were also used for the transgene expression (RT-qPCR) analyses. This allowed the simultaneous determination of Bt concentration and mRNA as a proxi of transgene activity. Between 5 to 10 mg of freeze-dried leaf material was ground using a FastPrep-24 Instrument (MP Biomedicals, Inc.) and homogenized in 1.5 ml of PBST-buffer (pH 7.4). After centrifugation the supernatants were diluted 1:100 with PBST-buffer. Standards were prepared using freeze-dried Cry1Ab toxin (M. Pusztai-Carey, Case Western Reserve University) similar to the Cry1Ab protein expressed in the MON810 maize plants. Twelve Cry1Ab concentrations were used for the calibration curve ranging from 0 to 4.4 ng/ml dissolved in PBST-buffer. The Cry1Ab concentration in the different genetic backgrounds was determined using the commercial double antibody sandwich (DAS) ELISA kit (Agdia®, USA). The standards were added to a 96-well ELISA microplate in duplicates, and the negative controls and the samples were added in triplicates. The optical density development was measured on a SPARK 10M multimode microplate reader (TECAN®) at 650 nm.
Das elisa kit
Manufactured by Agdia
Sourced in United States
The DAS-ELISA kit is a laboratory equipment used to detect and quantify the presence of specific target analytes in a sample. It is based on the enzyme-linked immunosorbent assay (ELISA) technique, which utilizes antibodies and color change to identify the analyte of interest.
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Lab products found in correlation
5 protocols using das elisa kit
1
Quantifying Bt Toxin Concentration in Leaves
2
SVNV Transmission by Soybean Thrips
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SVNV was maintained in the greenhouse on soybean plants with continual transmission by N. variabilis at temperature of 25 ± 1°C and a photoperiod of 16:8 (L:D) hours. Disease-free, V3 stage of soybean plants were alternately added into SVNV-infected colony at 4–5 week intervals, allowing inoculation by viruliferous soybean thrips for 3–4 weeks in 45.72 × 45.72 × 76.20 cm thrips-proof cage at which time they were used as viral source in experiments. Plants were routinely checked for symptom development which typically took 3 weeks. Leaf tissues were harvested from symptomatic plants and infection was confirmed using SVNV-specific double-antibody sandwich enzyme-linked immunosorbent assay, DAS-ELISA kit (Agdia, Elkhart, IN, United States).
3
Quantification of Bt Cry1Ab Protein in Maize Leaves
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The leaf samples originated from the same plants analysed for transgene expression. Approximately 10 mg of freeze-dried leaf material was ground using a FastPrep-24 Instrument (MP Biomedicals, Inc.) and homogenized in 1.5 ml of PBST-buffer (pH 7.4). After centrifugation the supernatants were diluted 1:50 with PBST-buffer. Standards were prepared using freeze-dried Cry1Ab toxin (M. Pusztai-Carey, Case Western Reserve University) identical with the Bt-protein expressed in the Bt maize plants. Thirteen Cry1Ab concentrations were used for the calibration curve ranging from 0 to 6.8 ng/ml dissolved in PBST-buffer. In addition 3 standards were prepared with control leaf extracts from conventional maize. The level of Bt protein in maize leaves was determined using the commercial double antibody sandwich (DAS) ELISA kit (Agdia). Standards and controls were added to a 96-well ELISA microplate in duplicates, samples were added in triplicates. The colour development was measured in a kinetic mode at 650 nm using a Bio-Tek Synergy HT multi detection microplate reader. The colour reaction was stopped after 16 minutes by adding 3 M sulphuric acid and colour intensity was read at 450 nm.
4
Detection of PNRSV in Cherry Leaves
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Field samples were tested for PNRSV with DAS-ELISA kit (Agdia Inc., Elkhart, IN, USA) following the manufacture’s procedure. Healthy cherry leaves were individually used as the negative control. The OD values of DAS-ELISA plates were recorded at 405 nm using an Thermo Multiskan Ascent. Once the OD value of sample/ that of negative control is ≥ 2, the sample is deemed as a positive sample.
5
Modified DAS-ELISA for MCMV Detection
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A commercial DAS-ELISA kit (Agdia) was used for MCMV detection, with some modification of the manufacturer's protocol. Briefly, 96-well plates were incubated overnight at 4°C with capture antibody (1:200 v/v with coating buffer) and then washed three times with phosphate-buffered saline containing Tween 20 (PBST, Agdia). Samples (100 µl per well), prepared as outlined below, were applied to two replicate wells, and the plates were incubated overnight at 4°C in a closed plastic box containing a moist paper towel. Plates were then washed seven times with PBST, incubated with the alkaline phosphatase enzyme conjugate for 1 h at 37°C, and washed six times before adding alkaline phosphatase substrate. The absorbance of samples at 405 nm (A 405 ) was determined at room temperature at 20 and/or 60 min after addition of substrate using a FilterMax F5 Multi-Mode Microplate reader (Molecular Devices, San Jose, CA). Samples were considered positive if the average sample absorbance was greater than twice the mean absorbance of healthy controls. General extraction buffer (GEB, Agdia) and extracts from leaves of MCMV-KS infected plants (Early Sunglow or Spirit) served as negative and positive controls for ELISA performance, respectively.
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