Attogene EL2051-01 Pentachlorophenol ELISA Kit Instruction Manual

Pentachlorophenol ELISA Kit
Competitive enzyme immunoassay kit for quantitative analysis of Pentachlorophenol
Catalog Number: EL2051-01
For Research Use Only. Not for use in Diagnostic Procedures.
Instruction Manual

Background

Pentachlorophenol belongs to the class of organochlorine pesticides and can be used as an insecticide, antibacterial agent, fungicide, algaecide, and preservative.  pentachlorophenol continues to be used, frequently in combination with other pesticides, and can easily accumulate in animals and can enter the human body through the food chain. Pentachlorophenol causes harmful effects on the liver, kidneys, blood, lungs, nervous system, immune system, and gastrointestinal tract and is a probable human carcinogen.
This kit is based on ELISA technology, which is fast, easy, accurate, and sensitive compared with common instrumental analysis and only needs 1.5 hours in one operation, it can considerably minimize operation error and work intensity.

Test Principle

This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigens. Pentachlorophenol residue in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme-labeled anti-antibody, TMB substrate is used to show the color. The absorbance of the sample is negatively related to the Pentachlorophenol residing in it, after comparing it with the Standard Curve, multiplied by the dilution factor, the Pentachlorophenol residue quantity in the sample can be calculated.

Applications

This kit can be used in quantitative and qualitative analysis of Pentachlorophenol residue in honey, milk, juice, and rice.

Cross Reactions

Pentachlorophenol ……………………………………………… 100%

Equipment and Reagents Needed (not provided)

5.1 Equipment

• ELISA reader (450nm/630nm)
• Rotary evaporator or nitrogen drying instruments
• Vortex mixer
• Timer
• Wash bottle
• Analytical balance
• Polystyrene centrifuge tube: 50ml, 2ml
• Micropipettes: 20μl-200μl, 100μl-1000μl
• 300μl-multipipette

5.2 Reagents

• Deionized water

Components Provided in This Kit

• Microtiter plate with 96 wells coated with antigen
• Standard solutions (5 bottles×0.8ml/tube) 0ppb, 0.2ppb, 0.6ppb, 1.8ppb, 5.4ppb, 8 . 1 p p b
• Spiking standard solution (0.8ml/tube) 100ppb
• Pentachlorophenol Antibody#1: 11mL
• 100X HRP-Conjugated Antibody#2: 0.25mL
• Antibody#2 Diluent: 20mL
• 20X Wash solution: 28mL
• TMB Substrate solution: 12mL
• Stop solution: 14mL

Reagents Preparation

• 1X Wash solution: combine one volume of the 20X Wash Solution with 19 volumes of deionized water. Mix well.
• 1X HRP-conjugated Antibody#2: combine one volume of the 100X HRP-Conjugated Antibody#2 with 99 volumes of Antibody#2 Diluent. Vortex for 10 seconds to mix. Prepare this solution fresh before each test.

Notice and Precautions Before Operation

• Please use one tip in the process of experiment and change the tips when absorbing different reagent.
• The stop solution is in hydrochloric acid, which is corrosive and an irritant. Avoid contact with skin and mucous membranes. Immediately clean up any spills and wash area with copious amounts of water. If contact should occur, immediately flush with copious amounts of water.
• If running more than two strips at once, the use of a multichannel pipette is required.
• Make sure that all experimental instruments are clean.
• Treated samples can be stored at 2-8o C for 24h in dark.

Assay Process

9.1 Instructions Prior to Beginning Assay

• Ensure that all reagents and microwells are at room temperature (20-25℃). Notice: The antibody solution should be stored at 4℃, which will be used immediately after taking out.
• Return all the rest reagents to 2-8℃ immediately after their use.
• Wash the microwells correctly; this is a vital factor in the reproducibility of the ELISA analysis.
• Avoid direct sunlight during the incubation; use the plate cover provided in the kit to cover the plate.

9.2 Steps in the Assay Process

1. Take all reagents out at room temperature (20-25℃) for more than 30min. Shake gently before use.
2. Get the microwells needed out and return the rest into the zip-lock bag at 2-8℃ immediately.
3. The diluted wash solution should be brought to room temperature before use.
4. Number every microwell position and all standards and samples should be run in duplicate. Record the standards and sample positions.
5. Dispense 50μL of the Pentachlorophenol standards, positive control, or sample into each well.
6. Dispense 100μL of Antibody#1 into appropriate test wells.
7. Shake the plate gently for 30 seconds using a back-and-forth motion.
8. Cover the plate. Incubate for 30 minutes at room temperature.
9. Decant the contents of the wells into an appropriate waste container.
10. Rinse the microwells with 250μL of the 1X wash solution for 3 times.
11. Absorb the residual water by inverting with absorbent paper to remove the last of the wash solution.
12. Add 150μL of freshly prepared 1X HRP-conjugated Antibody#2 to each well.
13. Shake the plate gently for 30 seconds using a back-and-forth motion.
14. Cover the plate. Incubate for 30 minutes at room temperature.
15. Decant the contents of the wells into an appropriate waste container.
16. Rinse the microwells with 250μL of the 1X wash solution for 3 times.
17. Absorb the residual water by inverting with absorbent paper to remove the last of the wash solution.
18. Add 100μL TMB substrate solution to each well, mix gently by shaking the plate manually and incubate for 5-15min at 25℃ with cover.
19. Add 100μL the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (Read the result within 5min after addition of stopsolution).

NOTE: It is also possible to measure by eye without stop solution if there is no ELISA reader.

Results

10.1 Calculating the Percentage absorbance

• The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied
  by 100%. Absorbance (%) = B / B0 *100
  B = the mean absorbance value of each standard or each sample
  B0 = absorbance value of zero standard

10.2 Drawing a Standard Curve

• To draw a standard curve, t he absorbance value of standards as the y-axis, semilogarithmic of the concentration of the standards (ppb) as the x-axis.
• The concentration of each sample (ppb), which can be read from the standard curve, is multiplied by the corresponding dilution factor of each sample followed, and the actual concentration of sample is obtained.

Sensitivity, Accuracy and Precision

11.1 Test Sensitivity:

• Overall Sensitivity ………………………………………. 0.2ppb

11.2 Detection limit:

• Milk/Juice/water …………………………………………………… 1ppb
• Honey ………………………………………………………………… 1ppb
• Rice/produce/processed food ………………………………….. 1ppb

11.3 Precision:

• C.V. of the ELISA kit ………………………………………… less than 10%

General Instructions

12.1 Temperature of Reagents and Samples

• The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature (20-25℃). The antibody solution should be stored at 4℃, which will be used immediately after taking out. If the antibody solution is return to room temperature before assay, the OD values will be higher, and the result of the assay will not be right.

12.2 Microwells

• Do not allow microwells to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tap the microwells holder.

12.3. Shaking of Reagents

• Shake each reagent gently before use.

12.4. Skin Protection

• Keep your skin away from the stop solution for it is the in HCl solution.

12.5 Out-of-Date Kits

• Don’t use kits that are out of date. Don’t exchange the reagents of different batches, or else it will drop the sensitivity.

12.6 General Comments

• Keep the ELISA kits at 2-8℃, do not freeze them. Seal rest micro well plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.
• 12.7 Special Issues Concerning Solutions and Reagents
• The substrate solution should be abandoned if it turns colors. The reagents may be turn bad if the absorbance value (450/630nm) of the zero standards is less than 0.5 (A450nm<0.5).

12.8 Special Issues Concerning Color

• The coloration reaction needs 30min after the addition of solution A and solution B, but you can prolong the incubation time ranges to 35min or more if the color is too light to be determined, never exceeding 40min, on the contrary, shorten the incubation time properly.

12.9 Incubation Temperatures

• Incubation temperature should be at room temperature (20-28℃). The higher or lower temperatures on day of testing will lead to experiment-to-experiment changes.

Storage

• Storage condition: 2-8℃
• Storage period: 12 months

About

Attorney is a biotechnology company located in Austin, Texas. Our focus is to enhance health and wellness by offering and developing customer-focused Life Science Products domestically and internationally. Our mission is to:

• Enhance detection technologies
• Enable rapid responses
• Enable impactful research discoveries

Attorney is a biotechnology company located in Austin, Texas. Our focus is to enhance health and wellness by offering and developing customer-focused Life Science Products domestically and internationally. Our mission is to:

• Enhance detection technologies
• Enable rapid responses
• Enable impactful research discoveries

Contact Us
3913 Todd Lane, Suite 310
Austin, Texas 78744
Phone:512-333-1330
Email:sales@attogene.com
Web:www.attogene.com
EL2051-01.V1_20220818

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