Mouse hemolysin (HMN) kit experimental detection method - Database & Sql Blog Articles

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Kaixin micro test

Test probe P100-M3

Mouse Hemolysin

(

HMN

)

Kit

Experimental Detection Method

This kit is for research use only. It is not intended for diagnostic or therapeutic purposes.

Experimental Principle

The Mouse Hemolysin (HMN) Kit determines the level of HMN in a sample using the double antibody sandwich method. A solid-phase antibody, which is a purified mouse hemolysin (HMN) antibody, is coated onto a microplate. The sample is then added to the microplate, followed by an HRP-labeled HMN antibody. This creates a complex of antibody-antigen-enzyme-labeled antibody. After washing, the substrate TMB is added for color development. TMB turns blue under the action of HRP and changes to yellow when acid is added. The intensity of the color correlates with the concentration of HMN in the sample. The absorbance at 450 nm is measured using a microplate reader, and the HMN concentration is calculated from a standard curve.

Kit Composition

1. 130x Washing Solution – 20ml × 1 bottle

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (720ng/L) – 0.5ml × 1 bottle

5. Enzyme-Labeled Coating Plate – 12 wells × 8

6. Sample Diluent – 6ml × 1 bottle

7. Color Development Agent A – 6ml × 1 bottle

8. Color Development Agent B – 6ml × 1 bottle

9. Standard Dilutions – 1.5ml × 1 bottle

10. Instructions – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If testing cannot be performed immediately, store samples at -20°C, avoiding repeated freeze-thaw cycles.

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

Kit Procedure

1. Standard Dilution: Prepare a series of standards from the original one. For example, dilute the 360 ng/L standard to 180 ng/L, 90 ng/L, etc., according to the provided chart.

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

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

4. Washing: Use 30x diluted washing solution. Wash 5 times, gently tapping to remove excess liquid.

5. Add Enzyme Label: Add 50 µl of enzyme-labeled reagent to each well except blanks.

6. Incubation: Repeat step 3.

7. Washing: Repeat step 5.

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

9. Stop Reaction: Add 50 µl of stop solution to each well to halt the reaction.

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

Calculation

Plot the standard curve using known concentrations and corresponding OD values. Determine the sample concentration based on its OD value and apply the dilution factor for accurate results.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme-labeled plates in a sealed bag.

2. If the washing solution crystallizes, warm it in a water bath before use.

3. Use a pipette for accuracy and keep loading time under 5 minutes.

4. Always include a standard curve and consider diluting high-concentration samples if necessary.

5. Do not reuse sealing films to prevent contamination.

6. Keep substrates away from light.

7. Follow instructions strictly and rely on microplate reader readings.

8. Treat all waste as biohazardous material.

9. Do not mix components from different batches.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

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