Swine encephalitis (JE) enzyme-linked immunoassay kit instructions - Database & Sql Blog Articles

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Swine Encephalitis (JE) Enzyme-Linked Immunosorbent Assay Kit

Instruction Manual

This kit is intended for research use only.

Experimental Principle

The level of Swine Encephalitis (JE) in the specimens is determined using a double antibody sandwich method. The microwell plate is coated with purified JE antibody to create a solid-phase antibody. The JE antigen is then added to the wells, followed by the addition of HRP-labeled JE antibody. This forms an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, TMB substrate is added, which turns blue under the catalysis of HRP and changes to yellow when acid is introduced. The intensity of the color is directly proportional to the JE concentration in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader, and the JE concentration is calculated from a standard curve.

Kit Composition

1. 30x Washing Solution – 20ml × 1 bottle
2. Stop Solution – 6ml × 1 bottle
3. Enzyme Standard Reagent – 6ml × 1 bottle
4. Standard (64pg/ml) – 0.5ml × 1 bottle
5. Enzyme Label Coating Plate – 12 wells × 8 strips
6. Standard Dilutions – 1.5ml × 1 bottle
7. Sample Diluent – 6ml × 1 bottle
8. Instructions – 1 copy
9. Reagent A – 6ml × 1 bottle
10. Color Developer B – 6ml × 1 bottle
11. Sealing Film – 2 sheets
12. Sealed Bag – 1

Sample Requirements

1. Specimens should be processed as soon as possible after collection, following relevant literature guidelines. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 cannot be used, as it inhibits HRP activity.

Procedure

1. Standard Dilution: Dilute the original standard according to the provided chart.
2. Loading: Set up blank, standard, and sample wells. Add 50μl standard, 40μl diluent, and 10μl sample per well.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash 5 times with diluted washing solution, then pat dry.
5. Add Enzyme: Add 50μl enzyme reagent to each well except blanks.
6. Incubation: Repeat step 3.
7. Color Development: Add 50μl developer and incubate at 37°C for 10 minutes.
8. Stop Reaction: Add 50μl stop solution to terminate the reaction.
9. Measurement: Read OD values at 450nm within 15 minutes.

Calculation

Plot a standard curve using standard concentrations vs. OD values. Determine the sample concentration from the curve or use linear regression for more accurate results. Multiply by the dilution factor to obtain the actual concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store unsealed enzyme reagents in a sealed bag.
2. Concentrated washing solution may crystallize; heat gently if needed.
3. Use a pipette for accuracy and avoid cross-contamination. Control loading time within 5 minutes.
4. Always make a standard curve and run duplicates. If OD exceeds the first standard, dilute the sample before testing.
5. Use the sealing film only once to prevent contamination.
6. Keep substrates away from light.
7. Follow instructions strictly and rely on microplate reader readings.
8. Treat all samples and waste as infectious materials.
9. Do not mix components from different batches.

Storage and Expiration

1. Store at 2–8°C.

2. Shelf life: 6 months.