Attogene EL2024-04 Congener Independent Microcystin ELISA Kit Instructions

Attogene EL2024-04 Congener Independent Microcystin ELISA Kit

Product Information

Specifications

• Product Name: Congener-Independent Microcystin ELISA Kit
• Type: Competitive enzyme immunoassay kit
• Analysis: Quantitative analysis of Microcystins and Nodularins
• Catalog Number: EL2024-04
• Intended Use: For Research Use Only. Not for use in Diagnostic Procedures.

Product Usage Instructions

Background
Microcystins are hepatotoxins produced by blue-green algae. This kit helps in the quantitative analysis of Microcystins and Nodularins.

Test Principle
This kit is a competitive enzyme immunoassay for the analysis of Microcystins and Nodularins.

Applications
The kit can be used for rapid testing of a broad spectrum of Microcystin congeners in liquid samples like water, wastewater, algal cultures, and solid samples like fish.

Sample Preparation

1. Liquid (water, wastewater, liquid media)
   Follow the provided guidelines for preparing liquid samples before conducting the assay.

2. Solid
   Prepare solid samples like fish as instructed in the manual before proceeding with the assay.

Background

Microcystins are a class of hepatotoxins produced by blue-green algae such as Microcystis aeruginosa. Microcystin-LR is the most common of the over 50 different congeners. Cyanobacteria can produce microcystin in large quantities during an algal bloom, which then poses a major threat to our aquatic ecosystems and sources of food, as there is a well-documented phenomenon called “bioaccumulation” that describes the increase in consumed toxin quantities as trophic levels increase.

Test Principle

The Microcystin plate kit is a congener-independent competitive enzyme-labeled immunoassay. The Mi-crocystin sample extract and calibrators are pipetted into the test wells followed by the Microcystin antibody into the test wells to initiate the reaction. During the 30 minutes incubation period, Microcystin from the sample and Microcystin antigen compete for binding to the Microcystin antibody. The Microcystin antibody is captured on the walls of the test well. Following this 30-minute incubation, the contents of the wells are removed and the wells are washed to remove any unbound Microcystin and free Microcystin antibody. After wash, 1X HRP-conjugated Antibody#2 is added for 30 minutes incubation. The wells are washed afterwards, and a clear substrate is then added to the wells and any bound enzyme conjugate causes the conversion to a blue color. Following a 15-minute incubation, the reaction is stopped and the amount of color in each well is read. The color of the unknown samples is compared to the color of the calibrators and the Microcystin concentration of the samples is derived.

Applications

This kit can be used for rapid test of a broad spectrum of Microcystin congeners in liquid samples such as water, wastewater, algal cultures, and solid samples such as fish.

Cross Reactions

Equipment and Reagents Needed (not provided)

Equipment

• ELISA Reader (450nm/630nm)
• Deionized water
• Methanol
• Vortex mixer
• Timer
• Wash bottle
• Polystyrene centrifuge tube: 50mL, 2mL
• Micropipettes: 20μL-200μL, 100μL-1000μL
• 300μL-multipipette

Components Provided in This Kit

• Microtiter plate with 96 wells coated with Microcystin
• Microcystin (LR) Standards (6 vials × 0.8mL/vial): 0ppb (green cap), 0.05ppb (purple cap), 0.1ppb (yellow cap), 0.2ppb (blue cap), 0.4ppb (orange cap), 2ppb (red cap)
• Microcystin 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 Anti-body#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 a fresh tip in the process of experiment and change the tips when absorbing different reagent.
• 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 2-8℃ for 24 hours in the dark.

Sample Preparation

Liquid (water, wastewater, liquid media)

• Make sure sample is free of particles and adjusted to a neutral pH.
• If necessary, centrifuge to pellet insoluble material (3000g / 5 minutes / at room temperature or filter using a 1.2μm syringe filter).
• Take 50μL of the supernatant for assay.

Solid

• Homogenize with commercial blender for at least 3 minutes to be sure of homogeneity.
• Add 9.0 mL of 90% Methanol/water to 1 g of this homogenate and vortex for 2 minutes.
• Centrifuge to pellet insoluble material at 3000xg for 10 minutes.
• Dilute the supernatant with Antibody #2 Diluent.
• Use 50μL of the diluted supernatant for assay.

Assay Process

Instructions Prior to Beginning Assay

1. Ensure that all reagents and microwells are at room temperature (20-25℃).
2. Return all reagents to 2-8℃ immediately after their use.
3. Wash the microwells correctly; this is a vital factor in the reproducibility of the ELISA analysis.
4. Avoid direct sunlight during incubation.

Steps in the Assay Process

1. Take all reagents out at room temperature (20-25℃) for more than 30 minutes. 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 samples positions.
5. Dispense 50μL of Microcystin 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 3 times.
11. Absorb the residual water by inverting with absorbent paper to remove the last of the Wash Solution.
12. Add 150μL of 1X HRP-Conjugated Antibody #2 (freshly prepared) 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 3 times.
17. Absorb the residual solution 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 15 minutes at 25℃ with cover.
19. Add 100μL of Stop Solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (Read the result within 5 minutes after addition of Stop Solution).

Results

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

Drawing a Standard Curve

• To draw a standard curve, the absorbance value of standards as y-axis, semilogarithmic of the concentration of the standards (ppb) as 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.
• Sample dilution factor: If the absorbance of a sample is lower than the highest calibrator (2 ppb), the concentration of Microcystin is too high and out of range of the standard curve. Dilute the sample and rerun. Samples should be diluted to fit into the standard curve (0.05 ppb to 2 ppb). Results must then be multiplied by the dilution factor used.

Sensitivity, Accuracy and Precision

1. Test Sensitivity:
   Overall Sensitivity …………………………………………………………………………………………….. 0.05ppb

2. Detection limit:
   Water, wastewater, culture media …………………………………………………………………… 0.05ppb

3. Accuracy:
   Water, wastewater, culture media …………………………………………………………………. 80±10%

4. Precision:
   C.V. of the ELISA kit ……………………………………………………………………………. less than 10%

General Instructions
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 restored to room temperature (20-25℃).

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 re stored to room temperature (20-25℃).

2. Microwells
   Do not allow microwell to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tapping the microwell holder.

3. Shaking of Reagents
   Shake each reagent gently before use.

4. Skin Protection
   The Stop Solution is 0.75N HCl, keep your skin away from it.

5. Out-of-Date Kits
   Don’t use kits that are expired. Don’t exchange the reagents of different batches, or else it will drop the sensitivity.

6. General Comments
   Keep the ELISA kits at 2-8℃, do not freeze. Store the unused microwell plates back to the foil pouch. Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.

7. Special Issues Concerning Solutions and Reagents
   The substrate solution should be abandoned if it turns color. The reagents may turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5 (A450nm<0.5).

8. Special Issues Concerning Color
   The colouration reaction takes 15 minutes after the addition of TMB Substrate, but you can prolong the incubation time ranges to 35 minutes or more if the color is too light to be determined, never exceed 40 minutes, on the contrary, shorten the incubation time properly.

9. Incubation Temperatures
   The incubation temperature should be at room temperature (20-25℃). Higher or lower temperatures on the day of testing will lead to experiment-to-experiment changes.

Storage

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

Customer Notes:

• Contact Us
  • Attorney 3913 Todd Lane, Suite 310 Austin, TX 78744
  • Phone: 512-333-1330
  • Email: sales@attogene.com
  • Web: www.attogene.com

FAQ

1. Can this kit be used for diagnostic procedures?
No, this kit is for research use only and is not intended for diagnostic procedures.

2. What is the importance of washing the microwells correctly?
Proper washing of microwells is crucial for the reproducibility of the ELISA analysis.

References

• Home Update - Attogene
• Home Update - Attogene

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