Insect trehalose ELISA kit technical guidance - Database & Sql Blog Articles

Insect Trehalose

ELISA

Kit

Technical Support

Experimental Principle

Insect Trehalose

ELISA

Kit

The level of insect trehalose in the sample is determined using a double-antibody sandwich ELISA method. A solid-phase antibody, purified insect trehalose antibody, is coated on a microplate. The sample containing trehalose is added and binds to the immobilized antibody. Then, an HRP-labeled trehalose antibody is introduced, forming an antibody-antigen-enzyme-labeled antibody complex. After washing, the substrate TMB is added. Under the catalytic action of HRP, TMB turns blue and then yellow upon acid addition. The intensity of the color correlates with the trehalose concentration in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader, and the sample concentration is calculated based on a standard curve.

Insect Trehalose

ELISA

Kit

Composition

130x Washing Solution – 20ml × 1 bottle | 7 Stop Solution – 6ml × 1 bottle

2 Enzyme Standard Reagent – 6ml × 1 bottle | 8 Standard (800ng/L) – 0.5ml × 1 bottle

3 Enzyme Labelled Coating Plate – 12 wells × 8 | 9 Standard Dilutions – 1.5ml × 1 bottle

4 Sample Diluent – 6ml × 1 bottle | 10 Instructions – 1 copy

5 Color Developer A – 6ml × 1 bottle | 11 Sealing Film – 2 sheets

6 Color 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. Perform the experiment promptly after extraction. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 cannot be used, as it inhibits horseradish peroxidase (HRP) activity.

Insect Trehalose

ELISA

Kit

Procedure

1. Standard Dilution: One original standard is provided. Prepare standards by diluting according to the chart below:

400ng/L No.5 – 150μl original + 150μl diluent

200ng/L No.4 – 150μl No.5 + 150μl diluent

100ng/L No.3 – 150μl No.4 + 150μl diluent

50ng/L No.2 – 150μl No.3 + 150μl diluent

25ng/L No.1 – 25μl No.2 + 25μl diluent

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

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

4. Washing: Use 30x diluted washing solution. Wash 5 times, then pat dry.

5. Add Enzyme: Add 50μl enzyme reagent to all wells except blanks.

6. Incubation: Repeat step 3.

7. Washing: Repeat step 4.

8. Color Development: Add 50μl developer A and B, incubate at 37°C for 10 minutes.

9. Stop Reaction: Add 50μl stop solution to each well.

10. Measurement: Read OD at 450nm within 15 minutes of adding stop solution.

Calculation

Plot a standard curve using standard concentrations vs. OD values. Determine the sample concentration from the curve, multiply by the dilution factor. Alternatively, use linear regression to calculate the sample concentration.

Insect Trehalose

ELISA

Kit

Precautions

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

2. Wash solution may crystallize; dissolve in warm water if needed. It won’t affect results.

3. Use a pipette for accuracy. Keep loading time under 5 min. For large samples, use a pipetting gun.

4. Always make a standard curve and run duplicates. If sample OD exceeds first standard, dilute the sample and adjust calculations accordingly.

5. Use one sealing film per test to prevent cross-contamination.

6. Keep substrates away from light.

7. Follow instructions strictly. Results must be confirmed by microplate reader.

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

9. Do not mix components from different batches.

Insect Trehalose

ELISA

Kit

Storage and Shelf Life

1. Store at 2–8°C.

2. Shelf life: 6 months.