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

(Enzyme-Linked Immunosorbent Assay)

Product No. 125011

Product Name

Generic Name: Microcystin-LR (MC-LR) Analysis Kit (Enzyme-Linked Immunosorbent Assay)

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

Enzyme-linked immunosorbent assay (ELISA) is a type of immunoenzymatic technique that combines the specificity of antigen-antibody reactions with the sensitivity of enzymatic reactions, establishing a novel methodology.

The technical principle of ELISA is as follows: Enzyme molecules are conjugated with antibodies (or antigens) to form stable enzyme-labeled antibody (or antigen) complexes. When these enzyme-labeled antibodies (or antigens) bind to their corresponding antigens (or antibodies) immobilized on a solid-phase carrier, a visible color reaction occurs in the presence of a substrate solution. The intensity of the color is proportional to the amount of antigen or antibody present. Quantitative analysis can be performed by measuring the absorbance using an ELISA reader, also known as a microplate reader.

This technique offers advantages such as high specificity, sensitivity, objective result interpretation, simplicity, and safety. Consequently, it has gained increasing attention and is widely applied not only in microbiology but also in various other scientific disciplines.

The assay exhibits very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date.

The working principle of this kit is illustrated in the following diagram.

Kit Components

The ELISA kit includes the following components:

1. Pre-coated 96-well microplate
2. Standard 1: MC-LR standard solution (0.1 ng/mL)
3. Standard 2: MC-LR standard solution (0.2 ng/mL)
4. Standard 3: MC-LR standard solution (0.5 ng/mL)
5. Standard 4: MC-LR standard solution (1 ng/mL)
6. Standard 5: MC-LR standard solution (2 ng/mL)
7. 1 vial of Solution 1 (Monoclonal antibody)
8. 1 tube of Solution 2 (For secondary antibody dilution only. Note: This is not the secondary antibody solution. Please measure according to the dilution ratio!)
9. Secondary antibody (The tube contains 2.4 μL enzyme-labeled secondary antibody. Store at 4°C)
10. 1 vial of Solution 3 (Substrate solution)
11. 1 vial of Solution 4 (Stop solution)
12. 1 packet of PBS powder (For washing buffer preparation)
13. 1 tube of Tween 20 (For washing buffer preparation)

Required Equipment and Reagents (Not Provided)

1. Microplate reader (preferably with plate washer if available)

2. Micropipettes (100 μL) (multi-channel pipettes [8 or 12 channels] recommended if available)

3. Pipettes and rubber pipette tips (for plate washing – not required if using plate washer)

4. Deionized water (for washing buffer preparation)

5. Glass bottles (for washing buffer storage)

6. Timer (for precise control of incubation steps)

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.

Preparation of Enzyme-Labeled Secondary Antibody

Dilute the secondary antibody of the tubule with Solution 2 at a ratio of 1:5000, mix thoroughly until completely dissolved, and prepare fresh for immediate use.

Preparation of washing buffer

Prepare 1 L of PBS solution (pH 7.4–7.6) by dissolving powdered PBS in Deionized water. Then, add 0.5 mL of Tween 20 into the prepared PBS solution. Store at room temperature. 

Test Procedure

1. Sample and Antibody Incubation

Add 50 μL of toxin standard or sample to each corresponding well (refer to Table 1), followed by 50 μL of monoclonal antibody (Solution 1) to each well. Mix gently and incubate at 37°C or room temperature for 45-90 minutes.  

Note: The standard/sample must be added before the monoclonal antibody solution.

2. Plate Washing (Manual)

Vigorously flick the plate over a sink to remove liquid. Using a pipette, fill each well completely with wash buffer (avoid overflow). Flick out wash buffer and repeat this wash procedure three times (3 minutes per wash). Blot dry on filter paper.  

Note: Automated plate washers may be used if available. Avoid cross-contamination between wells!

3. Enzyme-Conjugated Secondary Antibody Incubation

Add 100 μL of diluted enzyme-labeled secondary antibody to each well. Incubate at 37°C or room temperature for 30 minutes.

4. Plate Washing

Repeat washing procedure as in Step 2, but perform five washes (3 minutes each).

5. Substrate (Solution 3) Addition

Using a multichannel pipette or rubber pipette tips, immediately add 100 μL of substrate solution to each well. Incubate at 37°C or room temperature for color development. Monitor frequently; development should not exceed 30 minutes.

6. Reaction Termination

Upon visible color development, immediately terminate the reaction by adding 50 μL of Solution 4 (1 mol/L H₂SO₄).

7. Result Interpretation

Visual inspection: Negative control wells should show distinct yellow coloration against white background.  

Spectrophotometric measurement: Read absorbance at 450 nm using a microplate reader.  

Notes: All measurements must be completed within 30 minutes after termination.

Note: Precise pipetting is essential. Avoid cross-well contamination by ensuring no reagent spillover occurs between wells. Use dedicated pipette tips for each reagent to prevent cross-contamination.

Evaluation

1. ?Microplate Well Working Scheme

  C1、C2、C3、C4、C5： Standards；  S1~S42：Samples

2. Calculate the mean OD₄₅₀ value for each standard or sample, then determine B₀ using the following formula:

B₀ =   OD _{Standard or Sample}

                   OD _{Negative control}

• Generate the standard curve by plotting the logarithms of standard concentrations against their corresponding B₀?values. The standard curve shown in the following figure may serve as a reference.

• Determine sample concentrations by extrapolation from the standard curve.

NOTE: If a sample’s B falls outside the standard range (below 0.1 ng/mL or above 2.0 ng/mL), concentrate or dilute the sample accordingly and retest.

Institute of Hydrobiology,