产品编号:L48200451-096
产品描述:Triclosan ELISA kit(三氯生检测试剂盒)
反应种属:
实验方法:
标记:
规格:96wells
供应商:Biosense
价格(RMB):17475.00
订购:
说明书:
Triclosan ELISA kit
• Intended Use
For detection of Triclosan and Triclosan methyl. Please refer to the attached specific proceduresfor water (groundwater, surface water, well water,effluent), and soil. Application procedures for other sample matrices can be obtained from Abraxis.
• Principle
The Abraxis Triclosan Microtiter Plate Kit applies the principles of enzyme linked immunosorbent assay (ELISA) to the determination of Triclosan. In the assay system, standards, controls, or samples are added, along with an antibody specific to Triclosan, to microtiter wells coated with Goat Anti-Rabbit Antibody and incubated for thirty (30) minutes. The Triclosan enzyme conjugate is then added. At this point, a competitive reaction occurs between the Triclosan, which may be in the sample, and the enzyme-labeled Triclosan analog for the antibody binding sites on the microtiter well. The reaction is allowed to continue for thirty (30) minutes. After a washing step, the presence of Triclosan is detected by adding the “Color Solution,” which contains the enzyme substrate (hydrogen peroxide) and the chromogen (3,3’,5,5’- tetramethylbenzidine). The enzyme-labeled Triclosan bound to the Triclosan antibody catalyzes the conversion of the substrate/chromogen mixture to a colored product. The color reaction is stopped and stabilized after a twenty (20) minute incubation period by the addition of diluted acid (stopping solution). The color is then evaluated using an ELISA reader.
A dose response curve of absorbance vs. concentration is generated using results obtained from the standards. The concentration of Triclosan present in the control and samples is determined directly from this curve. Since the labeled Triclosan (conjugate) was in competition with the unlabeled Triclosan (sample) for the antibody sites, the intensity of the color developed is inversely proportional to the concentration of Triclosan present in the sample.
• Reagents
The Abraxis Triclosan Plate Kit contains the
following items:
1. Microtiter Plate coated with Goat-Anti Rabbit Antibody
96 test kit: 12 strips of 8 antibody
coated wells and strip holder (1).
2. Triclosan Antibody Solution
Triclosan antibody (rabbit anti-Triclosan) solution in a colored (red) buffered saline solution with preservative and stabilizers.
96 test kit: One vial containing 6 mL
3. Triclosan Enzyme Conjugate
Horseradish peroxidase (HRP) labeled Triclosan analog in a colored (green) buffered solution with preservative and stabilizers.
96 test kit: One vial containing 6 mL
4. Triclosan Standards
Six concentrations (0.05, 0.1, 0.25, 0.5, 1.0, 2.5 ppb) of Triclosan standards in distilled water with preservative and stabilizers.
96 test kit: Each vial contains 1.0 mL
5. Control
A concentration (approximately 0.75 ppb) of Triclosan in distilled water with preservative and stabilizers.
96 test kit: One vial containing 1.0 mL
6. Diluent/Zero Standard (Sample Diluent)
Distilled water with preservative and stabilizers without any detectable Triclosan.
96 test kit: One bottle containing 30 mL
7. Color Solution
A solution of hydrogen peroxide and 3,3',5,5'-tetramethyl benzidine in an organic base.
96 test kit: One bottle containing 16 mL
8. Stopping Solution
A solution of diluted acid.
96 test kit: Two bottles containing 6 mL each
9. Washing Buffer (5x) Concentrate
Buffered salts with detergent and preservatives.
96 test kit: One bottle containing 100 mL
• Reagent Storage and Stability
Store all reagents at 2-8°C. Do not freeze. Reagents may be used until the expiration date on the box.
Consult state, local and federal regulations for proper disposal of all reagents.
• Materials Required but Not Provided
In addition to the reagents provided, the following items are essential for the performance of the test:
Precision pipets capable of delivering 50, 100 and 250 uL, and tips*
Tape or Parafilm®*
Timer*
Distilled or deionized water for diluting Wash Buffer
Storage bottle with 1000 mL capacity for storage of 1x Wash Buffer*
Microplate or strip reader capable of reading absorbance at 450 nm*
l Please contact Abraxis for supplier information.
• Sample Information
This procedure is recommended for use with water samples. Other samples may require modifications to the procedure and should be thoroughly validated.
Samples containing gross particulate matter should be filtered (e.g. 0.2 um Anotop™25 Plus, Whatman, Inc.) to remove particles.
Samples which have been preserved with monochloroacetic acid or other acids, should be neutralized with strong base e.g. 6N NaOH, prior to assay.
If the Triclosan concentration of a sample exceeds 2.5 ppb, the sample is subject to repeat testing using a diluted sample. A ten-fold or greater dilution of the sample is recommended with an appropriate amount of Diluent/ZeroStandard or Sample Diluent. For example, in aseparate test tube make a ten-fold dilution byadding 100 uL of the sample to 900 uL ofDiluent/Zero Standard. Mix thoroughly beforeassaying. Perform the assay according to theAssay Procedure and obtain final results bymultiplying the value obtain by the dilution factor,e.g. 10.
The presence of the following substances up to 1,000 ppm were found to have no significant effect on the Triclosan Plate Assay results: phosphate, sodium fluoride, sodium chloride, magnesium and humic acid. Nitrate, manganese, calcium, sodium thiosulfate and sulfate up to 10,000 ppm. Copper, iron and zinc up to 100 ppm.
• Reagent Preparation
All reagents must be allowed to come to room temperature.
Wash Buffer
In a 1000 mL container, dilute the wash buffer concentrate 1:5 by the addition of distilled or deionized water (i.e., 100 mL of wash buffer concentrate plus 400 mL of H2O). This solution is used to wash the antibody coated wells.
• Procedural Notes and Precautions
As with all immunoassays, a consistent technique is the key to optimal performance. To obtain the greatest precision, be sure to treat each well in an identical manner.
Add reagents directly to the bottom of the well while avoiding contact between the reagents and the pipet tip. This will help assure consistent quantities of reagent in the test mixture.
Avoid cross-contaminations and carryover of reagents by using clean pipets for each sample addition and by avoiding contact between reagent droplets on the tubes and pipet tips.
The microtiter plate consists of 12 strips of 8 wells. If fewer than twelve strips are used, remove the unneeded strips and store refrigerated in the resealable foil bag (with desiccant) provided.
If more than 3 strips are being used per run, the use of a multi-channel pipette is recommended for the addition of conjugate, antibody, color, and stopping solutions.
Do not use any reagents beyond their stated shelf life. Each component used in any one assay should be of the same lot number and stored under identical conditions.
Avoid contact of Stopping Solution (diluted sulfuric acid) with skin and mucous membranes. If this reagent comes in contact with skin, wash with water.
• Limitations
The Abraxis Triclosan Plate Assay will detect Triclosan, Triclosan methyl and related compounds. Refer to the specificity table for data on several related compounds. The Abraxis Triclosan Plate Assay kit provides screening results. As with any analytical technique (GC, HPLC, etc...) positive results requiring some action should be confirmed by an alternative method.
• Quality Control
A control solution at approximately 0.75 ppb of Triclosan is provided with the Abraxis Triclosan Plate Assay kit. It is recommended that it be included in every run and treated in the same manner as unknown samples. Acceptable limits should be established by each laboratory.
• Assay Procedure
Read Reagent Preparation, Procedural Notes and Precautions before proceeding.
St0-St6: Standards
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1. Add 50 uL of the appropriate standard, control, or sample. Analysis in duplicates or triplicates is recommended.
2. Add 50 uL of Triclosan antibody solution successively to each well. Cover wells with parafilm or tape to prevent contamination and evaporation. Thoroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20- 30 seconds. Be careful not to spill the contents. Incubate at ambient temperature for 30 minutes.
3. After the incubation, add 50 uL of Triclosan enzyme conjugate solution successively to each well. Cover wells with parafilm or tape and thoroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Incubate at ambient temperature for 30 minutes.
4. After the incubation, carefully remove the covering and vigorously shake the contents of the wells into a waste container. Wash the strips with the diluted Wash Buffer (see Reagent Preparation) by adding a volume of at least 250 uL of Wash Buffer to each well. Vigorously shake the contents of the wells into a waste container. Any remaining buffer in the wells should be removed by patting the plate on a dry stack of paper towels. Repeat this wash step two times, for a total of 3 rinses.
5. Add 100 uL of Color Solution successively to each well. Cover wells with parafilm or tape. Thoroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds Incubate at ambient temperature for 20 minutes.
6. Add 50 uL of Stopping Solution successively to each well.
7. Read absorbance using a microplate reader at 450 nm within 15 minutes after adding the Stopping Solution.
• Results
The evaluation of the ELISA can be performed using commercial ELISA evaluation programs (4- parameter or alternatively point to point). For 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 each standard by the mean absorbance value for the Diluent/Zero Standard (Standard 0). Construct a standard curve by plotting the %B/Bo for each standard on the vertical linear (Y) axis versus the corresponding Triclosan concentration on the horizontal log (X) axis on the graph paper provided. Calculate the %B/Bo for the control and sample(s) and obtain the concentration of Triclosan (in ppb) by interpolation using the constructed standard curve.
Samples exhibiting a concentration lower than 0.020 ppb should be assumed to be below the detection limit of the assay. Samples exhibiting a concentration higher than 2.5 ppb must be diluted to obtain accurate results.
• Performance Data
Precision
The following results were obtained:
Control 1 2 3
Replicates 5 5 5
Days 5 5 5
n 25 25 25
Mean (ppb) 0.097 0.236 0.926
% CV (within assay) 9.7 7.3 8.3
% CV (between assay) 12.4 12.4 9.6
Limit of Detection
The Abraxis Triclosan Plate Assay has an estimated minimum detection concentration based on a 90% B/Bo of 0.020 parts per billion (ppb).
Recovery
Four (4) groundwater samples were spiked with various levels of Triclosan and then assayed using the Abraxis Triclosan Plate Assay. The following results were obtained:
Amount of -------------- Recovery -------------
Triclosan Mean S.D.
Added (ppb) (ppb) (ppb) %
0.5 0.467 0.029 93
1 1.122 0.079 112
2 2.146 0.082 107
Average
Sensitivity
The Abraxis Triclosan Plate Assay has an estimated minimum detectable concentration, based on a 90% B/Bo of 20 ppt (0.020 ppb). Refer to appropriate application notes or procedures for sensitivity in specific sample matrices.
Specificity
The cross-reactivity of the Abraxis Triclosan Plate Assay for various related and unrelated compounds can be expressed as the least detectable dose (LDD) which is estimated at 90% B/Bo, or as the dose required to displace 50% (50% B/Bo).
LDD 50%
B/Bo Compound (ppb) (ppb)
Triclosan 0.020 0.250
Triclosan methyl 0.015 0.080
PBDE Congener 28 0.034 0.61
PBDE Congener 47 0.020 0.390
PBDE Congener 49 5.2 17.8
PBDE Congener 99 2.15 15.0
4’-OH-BDE-47 0.13 7.8
5-OH-BDE-47 0.15 5.6
6-OH-BDE-47 0.66 10.2
2,4,5-Tribromobiphenyl >100 >100
2,4’,5-Tribromobiphenyl 54 9,100
2,3,7,8-Tetrachloro-dibenzo-p-dioxin >100 >100
T3 0.94 40
L-Thyroxine (T4) 340 700
The following compounds demonstrated no reactivity in the Triclosan Plate Assay at concentrations up to 1,000 ppb: Biphenyl, 2,4-D.
• Assistance
For ordering or technical assistance contact:
Abraxis LLC
Sales Department
54 Steamwhistle Drive
Warminster, PA 18974
(215) 357-3911 * Fax (215) 357-5232
• Ordering Information
Abraxis Triclosan Assay Kit, 96T PN 530114
Triclosan Sample Diluent PN 530112
• General Limited Warranty
Abraxis LLC warrants the products manufactured by the Company, against defects and 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.
