Attogene EL2047-01 Cylindrospermopsin ELISA Kit Instructions

: June 9, 2024
: Attogene

Table of Contents

Attogene EL2047-01 Cylindrospermopsin ELISA Kit
Background
Test Principle
Specificity
Reagents Preparation
General Instructions
Safety
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Attogene EL2047-01 Cylindrospermopsin ELISA Kit

Background

The Cylindrospermopsin Plate Kit is a competitive ELISA for the quantitative analysis of Cylindrospermopsin in water.

Test Principle

Attogene’s Cylindrospermopsin ELISA Kit is a competitive enzyme-labeled immunoassay. Cylindrospermopsin- HRP enzyme conjugate is pipetted into the test wells followed by calibrators or samples. Cylindrospermopsin Antibody Solution is added into the test wells to initiate the reaction. During the 45-minute incubation period, Cylindrospermopsin from the sample and Cylindrospermopsin-HRP conjugate compete for binding to the cylindrospermopsin antibody. Following this incubation, the wells are washed to remove any unbound Cylindrospermopsin or Cylindrospermopsin-HRP conjugate. After washing, a colorless substrate is added to the wells and any bound Cylindrospermopsin- HRP conjugate will convert the substrate to a blue color. Following a 45-minute incubation, the reaction is stopped and the amount of color in each well is measured. The color of the unknown sample is compared to the color of the calibrators and the Cylindrospermopsin concentration of the sample is derived. The color intensity is inversely proportional to the amount of Cylindrospermopsin present in the sample.

Applications

This kit can be used for rapid test of Cylindrospermopsin in liquid samples such as water, wastewater, and algal cultures.

Components Provided in This Kit

Microtiter plate with 96 wells coated with antibody
Cylindrospermopsin standard solutions (5 bottles×2ml/bottle) 0ppb, 0.05ppb, 0.3ppb, 0.75ppb, 2.0ppb
Cylindrospermopsin Positive Control (0.25ppb) 2ml
Cylindrospermopsin enzyme conjugate 7ml
Cylindrospermopsin Antibody solution 7ml
Substrate solution 14ml
Stop solution 14ml

Equipment and Reagents Needed (not provided)

ELISA reader (450nm)
Deionized water
Vortex mixer
Timer
Wash bottle
Absorbent material
Micropipettes: 20μl-200μl, 100μl-1000μl
Multi-channel pipette: 8-channel (50 & 100 μl)

Specificity

Cylindrospermopsin residues can be detected by this assay. Common cyanotoxins which can be found in water samples were tested in the assay and their reactivity is listed in the table below.

Attogene-EL2047-01-Cylindrospermopsin-ELISA-Kit-FIG-1

Reagents Preparation

Solution 1: Wash solution

Dilute the 100X concentrated wash solution with deionized water in the volume ratio of 1:99, which will be used to rinse the plates. The diluted wash solution can be conserved for one month at 4℃.

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 1 N 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 2-8oC for 24h in dark.

Sample Preparation

Water samples should be free of particles and adjusted to a neutral pH.
If necessary, centrifuge or filter samples prior to running in the assay.

Assay Procedure

Bring all kit reagents and samples to room temperature.
Remove the required number of test wells from the re-sealable foil bag. Re-seal the remaining strips in the bag containing the desiccant to limit moisture exposure.
Dispense 50 μL of the HRP Enzyme Conjugate into each well.
Add 50 μL of the Calibrators, Positive Control or Samples into the appropriate wells. Be sure to use a clean pipette tip for each solution to avoid cross contamination.
Dispense 50 μL of the Antibody Solution into each well. Shake the plate gently to mix contents.
Incubate the wells for 45 minutes at room temperature.
After this incubation, decant the contents of the wells into an appropriate waste container. Flood the wells completely with laboratory grade water, then decant. Repeat this wash step three times for a total of four washes. Invert the plate on absorbent paper and tap out as much of the water wash solution as possible.
Add 100 μL of Substrate to each well. Shake the plate gently to mix contents.
Incubate the wells at room temperature for 45 minutes.
Add 100 μL of Stop Solution to each well. Shake the plate gently to mix contents.
Measure and record the absorbance (Optical Density; OD) of each well using a microtiter plate reader at 450nm. by shaking the plate manually and measure the absorbance at 450nm (Read the result within 5min after addition of stop solution).
If the absorbance of a sample is lower than the highest calibrator (2.0 ppb), the concentration of Cylindrospermopsin is too high and out of range of the standard curve. Dilute the sample in laboratory grade water and rerun. Samples should be diluted to fit into the standard curve (0.05 ppb to 2.0 ppb). Results must then be multiplied by the dilution factor used.
The value of the 0.25 ppb control should fall within the following range: 0.25 ppb Cylindrospermopsin Positive Control: 0.15 – 0.32 ppb

Results

Semi-quantitative results can be derived by simple comparison of the sample absorbance to the absorbance of the calibrator wells. Samples with lower absorbance (less color) than a calibrator well, have a concentration of Cylindrospermopsin greater than the concentration of the calibrator. Samples with higher absorbance (more color) than a calibrator well, have a concentration less than the concentration of the calibrator.
Quantitative interpretation requires graphing the absorbance (OD) from the calibrator wells (Y axis) versus the calibrator concentration (X axis). This can be done using a plate reader with software which uses either a 4-Parameter or Semi-log curve fit. If your plate reader software does not provide these curve fits, a spreadsheet that will perform the curve fit and sample concentration calculation is available upon request.

Attogene-EL2047-01-Cylindrospermopsin-ELISA-Kit-FIG-2

Actual values may vary; this data is for example purposes only.

Standard deviation
Bo% equals the average sample absorbance divided by the average 0 ppb Calibrator absorbance multiplied by 100.

General Instructions

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.

Microwells

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

Shaking of Reagents

Shake each reagent gently before use.

Skin Protection

Keep your skin away from the stop solution for it is the 1 N HCl solution.

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.

General Comments

Keep the ELISA kits at 2-8℃, do not freeze. Seal rest microwell plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.

Special Issues Concerning Solutions and Reagents

Substrate solution should be abandoned if it turns colors. The reagents may be turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5 (A450nm<0.5).

Incubation Temperatures

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

Safety

To receive complete safety information on this product, contact Attogene and request Safety Data Sheets. Stop Solution is 1N hydrochloric acid. Handle with care.

Who we are

Attogene 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