化学物質に関する簡易モニタリング技術 実証試験計画書
Contents
1.2. 10
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16. 999000 MoU 0 0 5 9 5 9 5 0 30 2 0 0 1 15 0 0
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22. Bana UBI 19 uuu uu uuu uuu Lu uu uu uu uuu Ug uuu LB uuu uuu 20 oO OC n o EmnD oo o o r3 nooo nooo noo noo nooo t ooo Ooo 1 L3 L3 L3 p3 L3 E3 EJ pE3 E3 E3 EJ EJ EJ E3 EJ ES p Jo o o S L3 C3 1 L3 r3 1 oOo oo oo oo oo L Ono Oo oo oo oo oo oo oo oOo Ez E oo oo oo oo 21 juu Lu Lu Lu umm LU Lu OU 0000 0000 ud
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25. 000000 n 10 a 0 x Sd 500a O 0S0 000 Sd Step 4 000000000000000 OOOO 5 uuu 6 uu 7 uu 8 uuu umm 9 umm uu uma OU umm stepe 00000000 Step 7 000 G 0 0 Detection Limit OOOOOOOOOO0O000000000000000000
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39. and the reagents to room temperature before use 2 Remove the number of microtiter plate strips required from the aluminum foil The remaining strips are stored back in the aluminum foil and closed again using the white plastic clip Store the remaining kit in the refrigerator 4 8 C 3 The standard solutions positive and negative controls enzyme conjugate substrate and stop solution are ready to use and do not require any further dilutions 4 Dilute the wash buffer ata ratio of 1 5 If using the entire bottle 100 mL then add to 400 mL of deionized or distilled water 5 The stop solution has to be handled with care as it contains diluted H2504 B Assay Procedure 1 Add 50 uL of the assay buffer into each individual well using a multi channel or stepping pipet 2 Add 25 uL of the standard solutions the controls or the samples into the wells of the test strips according to the working scheme given We recommend using duplicates or triplicates 3 Add 50 uL of enzyme conjugate solution to the individual wells successively using a multi channel pipette or a stepping pipette 4 Incubate the strips for 30 min at room temperature if possible use an orbital shaker 5 Wash the strips three times using the washing buffer solution Please use at least a volume of 300 uL of washing buffer for each well and each washing step Remaining buffer in the wells should be removed by patting the plate dry on a stock of paper 6 Add 100 uL of subs
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41. O 0000000000000000 3 16 Atrazine ELISA Microtiter Plate Enzyme Linked Immunosorbent Assay for the Determination of Atrazine in Water Samples Product No 520005 1 General Description The Atrazine ELISA is an immunoassay for the quantitative and sensitive detection of atrazine a triazine herbicide This test is suitable for the quantitative and or qualitative detection of atrazine in water samples A previous sample preparation is not required If necessary positive samples can be confirmed by HPLC GC MS or other conventional methods 2 Safety Instructions The standard solutions of the test kit contain the herbicide atrazine In addition the substrate solution contains tetramethylbenzidine and the stop solution contains diluted sulfuric acid Avoid contact of stopping solution with skin and mucous membranes If these reagents come in contact with the skin wash with water 3 Storage and Stability The atrazine ELISA should to be stored in the refrigerator 4 8 C The solutions have to be allowed to reach room temperature 20 25 C before use Reagents may be used until the expiration date on the box 4 TestPrinciple The test is based on the recognition of atrazine by specific antibodies Atrazine present in the sample and a triazine enzyme conjugate compete for the binding sites of the antibodies immobilized on the pl
42. ate After a washing step and addition of the substrate solution a color signal is produced The intensity of the blue color is inversely proportional to the concentration of the atrazine present in the sample The color reaction is stopped after a specified time and the color is evaluated using an ELISA reader 5 Limitations of the Atrazine ELISA Possible Test Interference Numerous organic and inorganic compounds commonly found in water samples have been tested and found not to interfere with this test However due to the high variability of compounds that might be found in water samples test interferences caused by matrix effects can t be completely excluded Mistakes in handling the test also can cause errors Possible sources for such errors can be Inadequate storage conditions of the test kit wrong pipetting sequence or inaccurate volumes of the reagents too long or too short incubation times during the immune and or substrate reaction extreme outside temperatures during the test performance lower than 10 C or higher than 30 C The Abraxis Atrazine ELISA kit provides screening results with any analytical technique GC HPLC etc positive samples requiring some action should be confirmed by an alternative method Importance of the Atrazine Determination Pesticides are frequently applied in agriculture to protect crops from pests and to protect the yield of the harvest However a part of the active substance doe
43. ay for the detection of atrazine based upon sheep antibodies Anal Lett 25 1992 1025 1037 4 B Hock T Giersch A Dankwardt K Kramer S Pullen Toxixity Assessment and On line monitoring Immunoassays Environ Toxicol Water Qual 9 1994 243 262 1o 0000 3 000 2 400 1 800 1 200 0 000 0 001 0 010 a 100 1 000 15 1 0 00000 DO000000000 00 0 0 0 9 B004 D 00 LLDI I U B B050 0 uuum LDD 50 B Bo Compound ppb ppb Atrazine 0 02 0 52 Propazine 0 01 0 45 Ametryn 0 11 50 Prometryn 0 35 60 Prometon 0 08 24 Desethyl Atrazine 2 0 21 Terbutryn 1 5 100 Simazine 0 28 4 5 Desisopropyl Atrazine 10 8 250 Cyanazine 0 21 16 2 Hydroxy Atrazine 0 36 680 1 JEC ATZ MG 20 0000 Five 5 groundwater samples were spiked with various levels of atrazine and then assayed using the Abraxis Atrazine Assay The following results were obtained Amount of Atrazine u dt Added ppb OOO ppb C U 0 5 0 52 105 1 0 1 02 102 2 0 1 98 99 4 0 3 45 86 Average 98 2 J EC ATZ MG
44. other than triazines have not been observed Samples Drinking water ground water and surface water were tested for matrix effects in the ELISA No matrix effects were determined Parallel determinations using HPLC and GC MS methods showed a good correlation in the atrazine concentrations found General Limited Warranty Abraxis LLC warrants the products manufactured by the Company against defectsand workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product s printed expiration date Abraxis makes no other warranty expressed or implied There is no warranty of merchantability or fitness for a particular purpose For ordering or technical assistance contact Abraxis LLC 54 Steamwhistle Drive Warminster PA 18974 Tel 215 357 3911 Fax 215 357 5232 Email info abraxiskits com WEB WWW abraxiskits com R112304 Working Instructions A Test Preparation Micro pipetting equipment and pipette tips for pipetting the standards and the samples are necessary We recommend using a multi channel pipette or a stepping pipette for adding the enzyme conjugate the substrate solution and the stop solution in order to equalize the incubations periods of the standard solutions and the samples on the entire microtiter plate Please only use the reagents and standards from one package lot in one test as they have been adjusted in combination 1 Adjust the microtiter plate
45. s not reach the target plant but evaporates during application or remains in the soil According to their wide application and the relatively high persistence they can be detected in rain surface water and in ground water The application of the herbicide atrazine is prohibited in several countries e g Germany In the U S according to the USEPA SWDA drinking water guidelines the MCL for atrazine in drinking water is not allowed to exceed 3 ppb Itis desirable to check water samples or food for possible residues of triazines as these herbicides frequently occur in water and soil The atrazine ELISA allows the determination of 40 samples in duplicate determination Only few mL of sample are required The test can be performed in less than 1 hour Performance Data Test sensitivity The detection limit for atrazine is 0 03ug L mean of 6 blank determinations minus 3 standard deviations The middle of the test 50 B Bo is at approximately 0 8 ug L Determinations close to the middle of the tests give the most accurate results Test reproducibility Coefficients of variation CVs for standards 1096 CVs for samples 1596 S electivity The ELISA for atrazine recognizes beside atrazine also propazine Cross reactivities atrazine 100 per definition Propazine 115 ametryn 1 4 deethylatrazine 2 5 hydroxyatrazine 0 01 cyanazine 3 3 simazine 11 6 prometon 2 2 prometryn 0 9 Cross reactivities with pesticide classes
46. tes with plastic tips 10 100 uL 100 1000 uL Multi channel pipette 10 200 uL or stepper pipette with plastic tips 10 200 uL Microtiter plate washer Microtiter plate reader wave length 450 nm Shaker for microtiter plates optional Ol W NF E Working Scheme The microtiter plate consists of 6 double strips which can be used individually for the test The standards have to be run with each test Never use the values of standards which have been determined in a test performed previously St0 St6 Standards 0 0 05 0 1 0 25 1 0 2 5 5 0 ppb NC Negative Control 0 03 ppb PC Positive Control 3 0 ppb Sal Sa2 533 etc Samples F Standard Curve These values are used for demonstration purposes do not use these values for your determinations 1 200 4 p 2 9 600 ioco ss lc ee DEPON T lm T a a 0 0041 3 G ino 1 000 10 09 References 1 A Dankwardt E M Thurman B Hock Terbuthylazine and deethylterbuthylazine in rain and surface water Determination by enzyme immunoassay and gas chromatography mass spectrometry Acta hydrochim hydrobiol 25 1997 5 10 2 A Dankwardt S Pullen S Rauchalles K Kramer F J ust B Hock Atrazine residues in soil two years after the atrazine ban A comparison of enzyme immunoassay with HP LC Anal Lett 28 1995 621 63 3 S W st B Hock A sensitive enzyme immunoass
47. trate solution to the wells The strips are incubated for 15 min at room temperature in the darkness if possible on a shaker Protect the strips from light Add 50 uL of stop solution to the wells in the same sequence as for the substrate solution Read the absorbance at 450 nm using a microplate ELISA photometer C Evaluation The evaluation of the ELISA can be performed using commercial ELISA evaluation programs Logit Log or 4 P arameter For a manual evaluation calculate the mean absorbance value for each of the standards Calculate the B Bo for each standard by dividing the mean absorbance value for the Zero Standard Standard 0 Construct a standard curve by plotting the B Bo for each standard on a vertical linear y axis versus the corresponding atrazine concentration on horizontal logarithmic x axis on graph paper B Bo for controls and samples will then yield levels in ppb of atrazine by interpolation using the standard curve The concentrations of the samples are determined using this standard curve Samples showing a lower concentrations of atrazine compared to standard 1 0 05 ug L are considered as negative Samples showing a higher concentration than standard 6 5 ug L must be diluted further to obtain more accurate results The concentration of the negative and positive controls should be in the range given in the test instructions 15 D Additional Materials not delivered with the test kit Micro pipet
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