(Time-Resolved Fluorescence Immunoassay)

Product No. 125033

Product Name

Generic Name: Microcystin-LR (MC-LR) Analysis Kit (Time-Resolved Fluorescence Immunoassay)

Packaging Specification

96 tests/kit

Intended Use

This kit is intended for the quantitative detection of microcystins in water samples, algal samples, and aquatic products.

Sample Preparation:

1. Water samples: Centrifuge the collected sample and use the supernatant or filter the sample and use the filtrate directly for testing.

2. Algal samples: Three processing options:

   – Repeated freeze-thaw (3–5 times)

   – Ultrasonic disruption

   – Cell press disruption

3. Aquatic products: Use published methods, e.g., Valéria Freitas de Magalh?es, Raquel Moraes Soares, Sandra M.F.O. Azevedo. Toxicon, 2001, 39:1077.

Principle of the Assay

Time-Resolved Fluorescence Immunoassay (TRFIA) is a new generation immunoassay technology known for its high sensitivity and stability. It uses lanthanide elements and their chelates with unique fluorescent properties as tracers to label antibodies or antigens, replacing enzymes, isotopes, or chemiluminescent substances. After antigen-antibody reactions, fluorescence intensity is measured by a time-resolved fluorescence analyzer.

In this kit, MC-LR in the sample competes with Europium (Eu)-labeled MC-LR-BSA for binding to monoclonal antibodies against MC-LR. The test wells are coated with goat anti-mouse IgG to capture the added mouse monoclonal antibodies. Then the sample and Eu-labeled MC-LR-BSA are added. After incubation and washing, enhancement solution is added to dissociate the Eu from the complex, producing strong fluorescence.

As all wells have the same number of antibody binding sites and the same amount of Eu-labeled MC-LR-BSA, samples with low MC-LR concentration bind more Eu-labeled MC-LR, resulting in higher fluorescence. Conversely, higher MC-LR concentrations result in lower fluorescence. Fluorescence intensity is inversely proportional to MC-LR concentration. The assay schematic for this kit is shown below:

Kit Components

Required Equipment and Reagents (Not Provided)

• Time-resolved fluorescence immunoassay analyzer
• 100 mL graduated cylinder or suitable container
• Oscillator
• Plate washer
• Micropipettes with various ranges
• Distilled or deionized water
• Other applicable reference standards

Storage Conditions and Shelf Life

1. Store at 2–8°C; shelf life: 12 months. After opening, store at 2–8°C and use within 4 weeks.
2. Unused wells should be resealed in the original foil pouch with desiccant.
3. See product label for manufacturing and expiration dates.

Applicable Instruments

1. DR6606 or DR6626 TRFIA analyzers (Guangzhou Darui?Biotechnology?Co., Ltd.)
2. Other TRFIA-compatible analyzers using lanthanide elements.

Test Procedure

1. Reagent Preparation
2. Wash Buffer: Dilute 40 mL of concentrated wash buffer with 1000 mL purified water (1:25).
3. Eu-labeled MC–LR-BSA: Dilute 1:20 with assay buffer. Prepare only as much as needed for the test.
4. Equilibrate coated plates to room temperature.
5. Add to each well: 50 μL MC-LR standard/sample → 50 μL Eu-labeled MC-LR-BSA → 50 μL MC-LR monoclonal antibody. Seal the plate and incubate at room temperature with shaking for 45 minutes.
6. Remove the covering, decant the contents of the wells into a sink, and blot the inverted plate on a stack of paper towels. Wash the wells three times using the diluted wash buffer.
7. Add 150 μL of enhancement solution to each well. Incubate at room temperature with shaking for 5 minutes.
8. Measure the fluorescence at 340 nm (excitation light)/ 615 nm?(emit?light) using the TRFIA analyzers.

Data Analysis

Two methods for standard curve generation and concentration calculation:

1. Semi-log Regression Method
2. After measurement, calculate the average fluorescence intensity of each standard. The fluorescence value of point A (0 ng/mL) is defined as B_max.
3. For standards B to F (0.1, 0.3, 1.0, 2.0, and 4.0 ng/mL), calculate their binding ratio (B) using the formula:?B?=?B_standard???/?B_max?
4. Then, plot the binding ratio (B) as?the Y-axis, and the natural logarithm of the standard concentrations (ln[MC]) as?the X-axis to generate a semi-logarithmic curve. Perform linear regression on this curve to obtain a fitting equation.
5. To determine the concentration of microcystins in unknown samples, calculate their binding ratio (B_sample?/ B_max) and substitute it into the fitted equation to interpolate the corresponding concentration.
6. Logit-Log Regression Method

The logit-log method is a classical model used for competitive immunoassays. It follows similar steps to the semi-logarithmic method:

• Calculate the average fluorescence intensity for each standard;
• Define the fluorescence value of point A (0 ng/mL) as B_max?;
• For standards B to F, calculate the binding ratio (B?=?B_standard???/?B_max?);
• Then, compute the logit value of B using: logit?(B) = ln[B/(1-B)], plot the logit?(B)?as the Y-axis, and the natural logarithm of the standard concentrations (ln[MC]) as the X-axis to generate a semi-logarithmic curve.
• To determine the concentration of microcystins in unknown?samples, calculate their binding ratio (B_sample?/ B_max) and substitute it into the fitted equation to interpolate the corresponding concentration.

NOTE: If a sample’s B falls outside the standard range (below 0.1 ng/mL or above 4.0 ng/mL), concentrate or dilute the sample accordingly and retest.

Performance Characteristics

1. Standard curve correlation coefficient (r2) ≥?0.99
2. Precision: The coefficient of variation (CV) of this kit should be ≤?15.0%

Precautions

1. Follow instructions strictly.
2. Do not mix reagents from different batches. Use all kit components once only.
3. Perform tests in a clean, dust-free environment.
4. Equilibrate all reagents and samples to room temperature before use.
5. Treat all samples as potentially infectious. Avoid contact with skin and mucous membranes.
6. Calibrate plate washer daily; ensure thorough washing and drying.
7. Use clean, disposable containers for Eu-labeled reagents to prevent contamination.
8. Avoid pipette tips touching reagents or well walls when adding enhancement solution.
9. Store all reagents at 2–8°C. Avoid repeated use of opened reagents. Reseal unused strips.

Instruction Manual for Microcystin-LR (MC-LR) Analysis Kit

(Time-Resolved Fluorescence Immunoassay)

Product No. 125033

Product Name

Generic Name: Microcystin-LR (MC-LR) Analysis Kit (Time-Resolved Fluorescence Immunoassay)

Packaging Specification

96 tests/kit

Intended Use

This kit is intended for the quantitative detection of microcystins in water samples, algal samples, and aquatic products.

Sample Preparation:

1. Water samples: Centrifuge the collected sample and use the supernatant or filter the sample and use the filtrate directly for testing.

2. Algal samples: Three processing options:

   – Repeated freeze-thaw (3–5 times)

   – Ultrasonic disruption

   – Cell press disruption

3. Aquatic products: Use published methods, e.g., Valéria Freitas de Magalh?es, Raquel Moraes Soares, Sandra M.F.O. Azevedo. Toxicon, 2001, 39:1077.

Principle of the Assay

Time-Resolved Fluorescence Immunoassay (TRFIA) is a new generation immunoassay technology known for its high sensitivity and stability. It uses lanthanide elements and their chelates with unique fluorescent properties as tracers to label antibodies or antigens, replacing enzymes, isotopes, or chemiluminescent substances. After antigen-antibody reactions, fluorescence intensity is measured by a time-resolved fluorescence analyzer.

In this kit, MC-LR in the sample competes with Europium (Eu)-labeled MC-LR-BSA for binding to monoclonal antibodies against MC-LR. The test wells are coated with goat anti-mouse IgG to capture the added mouse monoclonal antibodies. Then the sample and Eu-labeled MC-LR-BSA are added. After incubation and washing, enhancement solution is added to dissociate the Eu from the complex, producing strong fluorescence.

As all wells have the same number of antibody binding sites and the same amount of Eu-labeled MC-LR-BSA, samples with low MC-LR concentration bind more Eu-labeled MC-LR, resulting in higher fluorescence. Conversely, higher MC-LR concentrations result in lower fluorescence. Fluorescence intensity is inversely proportional to MC-LR concentration. The assay schematic for this kit is shown below:

Kit Components

Required Equipment and Reagents (Not Provided)

• Time-resolved fluorescence immunoassay analyzer
• 100 mL graduated cylinder or suitable container
• Oscillator
• Plate washer
• Micropipettes with various ranges
• Distilled or deionized water
• Other applicable reference standards

Storage Conditions and Shelf Life

1. Store at 2–8°C; shelf life: 12 months. After opening, store at 2–8°C and use within 4 weeks.
2. Unused wells should be resealed in the original foil pouch with desiccant.
3. See product label for manufacturing and expiration dates.

Applicable Instruments

1. DR6606 or DR6626 TRFIA analyzers (Guangzhou Darui?Biotechnology?Co., Ltd.)
2. Other TRFIA-compatible analyzers using lanthanide elements.

Test Procedure

1. Reagent Preparation
2. Wash Buffer: Dilute 40 mL of concentrated wash buffer with 1000 mL purified water (1:25).
3. Eu-labeled MC–LR-BSA: Dilute 1:20 with assay buffer. Prepare only as much as needed for the test.
4. Equilibrate coated plates to room temperature.
5. Add to each well: 50 μL MC-LR standard/sample → 50 μL Eu-labeled MC-LR-BSA → 50 μL MC-LR monoclonal antibody. Seal the plate and incubate at room temperature with shaking for 45 minutes.
6. Remove the covering, decant the contents of the wells into a sink, and blot the inverted plate on a stack of paper towels. Wash the wells three times using the diluted wash buffer.
7. Add 150 μL of enhancement solution to each well. Incubate at room temperature with shaking for 5 minutes.
8. Measure the fluorescence at 340 nm (excitation light)/ 615 nm?(emit?light) using the TRFIA analyzers.

Data Analysis

Two methods for standard curve generation and concentration calculation:

1. Semi-log Regression Method
2. After measurement, calculate the average fluorescence intensity of each standard. The fluorescence value of point A (0 ng/mL) is defined as B_max.
3. For standards B to F (0.1, 0.3, 1.0, 2.0, and 4.0 ng/mL), calculate their binding ratio (B) using the formula:?B?=?B_standard???/?B_max?
4. Then, plot the binding ratio (B) as?the Y-axis, and the natural logarithm of the standard concentrations (ln[MC]) as?the X-axis to generate a semi-logarithmic curve. Perform linear regression on this curve to obtain a fitting equation.
5. To determine the concentration of microcystins in unknown samples, calculate their binding ratio (B_sample?/ B_max) and substitute it into the fitted equation to interpolate the corresponding concentration.
6. Logit-Log Regression Method

The logit-log method is a classical model used for competitive immunoassays. It follows similar steps to the semi-logarithmic method:

• Calculate the average fluorescence intensity for each standard;
• Define the fluorescence value of point A (0 ng/mL) as B_max?;
• For standards B to F, calculate the binding ratio (B?=?B_standard???/?B_max?);
• Then, compute the logit value of B using: logit?(B) = ln[B/(1-B)], plot the logit?(B)?as the Y-axis, and the natural logarithm of the standard concentrations (ln[MC]) as the X-axis to generate a semi-logarithmic curve.
• To determine the concentration of microcystins in unknown?samples, calculate their binding ratio (B_sample?/ B_max) and substitute it into the fitted equation to interpolate the corresponding concentration.

NOTE: If a sample’s B falls outside the standard range (below 0.1 ng/mL or above 4.0 ng/mL), concentrate or dilute the sample accordingly and retest.

Performance Characteristics

1. Standard curve correlation coefficient (r2) ≥?0.99
2. Precision: The coefficient of variation (CV) of this kit should be ≤?15.0%

Precautions

1. Follow instructions strictly.
2. Do not mix reagents from different batches. Use all kit components once only.
3. Perform tests in a clean, dust-free environment.
4. Equilibrate all reagents and samples to room temperature before use.
5. Treat all samples as potentially infectious. Avoid contact with skin and mucous membranes.
6. Calibrate plate washer daily; ensure thorough washing and drying.
7. Use clean, disposable containers for Eu-labeled reagents to prevent contamination.
8. Avoid pipette tips touching reagents or well walls when adding enhancement solution.
9. Store all reagents at 2–8°C. Avoid repeated use of opened reagents. Reseal unused strips.