Attogene EL2047-01 Operating instructions

Brand: Attogene

Model: EL2047-01

Type: Operating instructions

Attogene EL2047-01 is a competitive ELISA kit designed for the quantitative analysis of Cylindrospermopsin in water samples. It utilizes a competitive enzyme-linked immunoassay to detect Cylindrospermopsin with a detection range of 0.05 to 2.0 ppb. The kit includes all necessary reagents and standards for accurate and reliable testing.

Cylindrospermopsin ELISA Kit

Competitive enzyme immunoassay kit for

quantitative analysis of Cylindrospermopsin

Catalog Number: EL2047-01

For Research Use Only. Not for use in Diagnostic Procedures.

1. Background

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

in water.

2. 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.

3. Applications

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

and algal cultures.

4. 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

5. 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)

6. 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.

Compound

% Reactivity

Cylindrospermopsin

100%

7-Epi-Cylindrospermopsin

153%

7-Deoxy-Cylindrospermopsin

126%

Microcystin-LR

<1%

Nodularin

<1%

7. 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 conser ved

for one month at 4℃.

8. 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.

9. 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.

10. 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

11. 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.

Well Contents

Average OD± SD*

RSD%

**Bo%

0 ppb

2.15 ± 0.04

1.67

100

0.05 ppb

1.88 ± 0.02

1.23

0.3 ppb

0.51 ± 0.02

2.92

0.75 ppb

1.02 ± 0.02

3.07

2.00 ppb

0.23 ± 0.005

2.2

Positive Control

1.09 ± 0.03

2.4

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.

12. 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 temper-

ature on day of testing will lead to experiment-to-experiment changes.

13. 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:

• Enhance detection technologies

• Enable rapid responses

• Enable impactful research discoveries

3913 Todd Lane, Suite 310

Austin, TX 78744

Phone: 512- 333-1330

Email: [email protected]

Web: www.attogene.com

EL2047-01.V2_20220929

Attogene EL2047-01 Operating instructions

Attogene EL2047-01 Operating instructions

Attogene EL2047-01 Operating instructions

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