Plant folic acid (FA) ELISA kit operating instructions - Database & Sql Blog Articles

Plant Folic Acid (FA)

ELISA

Kit

Operating

Instruction Manual

Kit Instruction Manual

This kit is for research use only.

Experimental Principle

Plant Folic Acid (FA) ELISA Kit

The level of folate (FA) in plant samples is determined using a double-antibody sandwich ELISA. A microplate is coated with purified anti-plant folic acid (FA) antibodies to create a solid-phase antibody. Plant FA is then added to the wells, where it binds to the immobilized antibodies. Next, HRP-labeled anti-FA antibodies are introduced, forming an antigen-antibody-enzyme complex. After washing, TMB substrate is added, and the color changes from blue to yellow under the action of HRP and an acidic stop solution. The intensity of the color is directly proportional to the FA concentration in the sample. The absorbance (OD value) at 450 nm is measured using a microplate reader, and the FA concentration is calculated based on a standard curve.

Plant Folic Acid (FA) ELISA Kit - Composition

1. 30× Washing Solution – 20ml × 1 bottle

7. Stop Solution – 6ml × 1 bottle

2. Enzyme Standard Reagent – 6ml × 1 bottle

8. Standard (24μg/L) – 0.5ml × 1 bottle

3. Enzyme-Labeled Coating Plate – 12 wells × 8 strips

9. Standard Diluent – 1.5ml × 1 bottle

4. Sample Diluent – 6ml × 1 bottle

10. Instruction Manual – 1 copy

5. Reagent A – 6ml × 1 bottle

11. Sealing Film – 2 sheets

6. TMB Developer B – 6ml × 1 bottle

12. Sealed Bag – 1

Sample Requirements

1. Samples should be extracted as soon as possible after collection, following relevant literature procedures. If testing cannot be done immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 should not be used, as it may inhibit horseradish peroxidase (HRP) activity.

Plant Folic Acid (FA) ELISA Kit - Procedure

1. Standard Dilution: This kit includes one original standard. Prepare dilutions according to the following steps:

- Add 150 μl of original standard + 150 μl standard diluent → No. 5 standard (1 μl/L)

- Add 150 μl of No. 5 standard + 150 μl standard diluent → No. 4 standard (6 μg/L)

- Add 150 μl of No. 4 standard + 150 μl standard diluent → No. 3 standard (3 μg/L)

- Add 150 μl of No. 3 standard + 150 μl standard diluent → No. 2 standard (1.5 μg/L)

- Add 150 μl of No. 2 standard + 150 μl standard diluent → No. 1 standard (0.75 μg/L)

2. Loading: Set up blank, standard, and sample wells. Add 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample into each well (final dilution = 5x).

3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.

4. Washing: Dilute 30× washing solution with distilled water (1:30), and wash the plate 5 times.

5. Enzyme Addition: Add 50 μl of enzyme reagent to all wells except blank.

6. Incubation: Repeat step 3.

7. Color Development: Add 50 μl of TMB developer to each well, mix gently, and incubate at 37°C for 15 minutes.

8. Stop Reaction: Add 50 μl of stop solution to each well. The color will turn from blue to yellow.

9. Measurement: Read OD values at 450 nm within 15 minutes of adding the stop solution.

Calculation

Plot the standard curve using standard concentrations and corresponding OD values. Determine the sample concentration from the curve, multiply by the dilution factor. Alternatively, calculate the linear regression equation and use it to determine the sample concentration.

Precautions

1. Allow the kit to reach room temperature (15–30 min) before use. Store unopened enzyme reagents in sealed bags.

2. If the washing solution crystallizes, warm it in a water bath before use. It will not affect results.

3. Use accurate pipettes and calibrate frequently. Limit loading time to 5 minutes. For large numbers of samples, consider using an automated pipette.

4. Always prepare a standard curve with duplicate wells. If the sample OD exceeds that of the highest standard, dilute the sample before testing and adjust calculations accordingly.

5. Use the sealing film only once to avoid cross-contamination.

6. Keep the TMB substrate away from light.

7. Follow the manual instructions strictly. Rely on microplate reader readings for final results.

8. Treat all samples, washes, and waste as biohazardous materials.

9. Do not mix components from different batches.

10. In case of discrepancies, the English manual takes precedence.

Storage Conditions and Expiration Date

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.