Human lysozyme (LYS) ELISA kit instruction manual - Huaqiang Electronic Network

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Lysozyme (LYS)

ELISA

Reagent

Box instruction manual

This reagent is for research use only.

Purpose: This kit is used to determine the concentration of human serum, plasma, and related fluids.

Lysozyme (LYS)

content.

Experimental Principle:

The kit uses a double-antibody sandwich method. A monoclonal antibody against human lysozyme (LYS) is coated on a microplate. After adding the sample, the LYS binds to the immobilized antibody. Then, an HRP-labeled secondary antibody is added, forming an immune complex. The TMB substrate is then added, and the reaction produces a color change that is proportional to the LYS concentration. The final color is stopped with an acidic solution, turning blue to yellow. The absorbance at 450 nm is measured using a microplate reader, and the concentration is determined by comparing to a standard curve.

Kit Application:

Double Antibody Sandwich Method

Specimen:

Human Lysozyme (LYS) Level

Purified

Human Lysozyme (LYS) Antibody Coated Microplate

Anti-body, to the package

Monoclonal Antibody

Add in the micropores

Lysozyme (LYS)

Then bind to HRP-labeled Lysozyme (LYS) antibody to form antibody-antigen-enzyme-labeled antibody complex

After thorough washing

Plus

The substrate TMB developed color. TMB is at

HRP

The enzyme is converted to blue by catalysis and converted to the final yellow color by the action of an acid. The depth of the color and the sample

Lysozyme (LYS)

Positive correlation. Using a microplate reader

450

Absorbance (OD value) is measured at nm wavelength,

Passing the standard curve

Calculating samples

in

Human Lysozyme (LYS)

Content

.

Kit Composition:

Kit composition	48 hole configuration	96-well configuration
Instruction manual	1 copy	1 copy
Sealing film	2 pieces (48)	2 pieces (96)
Sealed bag	1	1
Enzyme label coated plate	1 ×48	1 ×96
Standard: 900 μg/L	0.5ml × 1 bottle	0.5ml × 1 bottle
Standard dilution	1.5ml × 1 bottle	1.5ml × 1 bottle
Enzyme standard reagent	3ml × 1 bottle	6ml × 1 bottle
Sample diluent	3ml × 1 bottle	6ml × 1 bottle
Developer A solution	3ml × 1 bottle	6ml × 1 bottle
Developer B solution	3ml × 1 bottle	6ml × 1 bottle
Stop solution	3ml × 1 bottle	6ml × 1 bottle
Concentrated washing solution	(20ml × 20 times) × 1 bottle	(20ml × 30 times) × 1 bottle

Sample Processing and Requirements:

1. Serum: Allow blood to clot naturally at room temperature for 10–20 minutes, then centrifuge for about 20 minutes (2000–3000 rpm). Carefully collect the supernatant. If precipitation occurs during storage, centrifuge again.

2. Plasma: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge for 20 minutes (2000–3000 rpm). Collect the supernatant carefully. If precipitate forms, centrifuge again.

3. Urine: Collect in a sterile tube and centrifuge for 20 minutes (2000–3000 rpm). Collect the supernatant carefully. If precipitate forms, centrifuge again. Similar procedure applies to pleural fluid and cerebrospinal fluid.

4. Cell culture supernatant: Collect in a sterile tube. Centrifuge for 20 minutes (2000–3000 rpm). For intracellular components, resuspend cells in PBS (pH 7.2–7.4), freeze-thaw repeatedly, and centrifuge again. Collect the supernatant carefully. If precipitate forms, centrifuge again.

5. Tissue specimen: Weigh the tissue, add PBS (pH 7.4), quickly freeze, and store in liquid nitrogen. After thawing, homogenize, centrifuge for 20 minutes (2000–3000 rpm), and collect the supernatant. Use part of the sample and freeze the rest.

6. Process specimens as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing.

7. Do not detect samples containing NaN3, as it inhibits horseradish peroxidase (HRP) activity.

Steps:

1. Dilute and load standards: Set up 10 standard holes on the enzyme-labeled plate. Add 100 µl of standard to the first and second wells, then add 50 µl of standard diluent and mix. Continue diluting serially down to the tenth well. Final concentrations: 600, 400, 200, 100, 50 µg/L.

2. Add samples: Set up blank wells (no sample or enzyme reagent). Add 40 µl of sample diluent to each sample well, then add 10 µl of sample (final dilution 5×).

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

4. Prepare washing solution: Dilute concentrated washing solution with distilled water (30 times for 48-well, 20 times for 96-well).

5. Wash: Remove the seal, discard liquid. Fill each well with washing solution, let stand for 30 seconds, discard, repeat 5 times, and pat dry.

6. Add enzyme reagent: Add 50 µl of enzyme-labeled reagent to each well (except blank wells).

7. Incubate: Repeat step 3.

8. Wash: Repeat step 5.

9. Color development: Add 50 µl of developer A, then 50 µl of developer B. Shake gently and incubate in the dark at 37°C for 15 minutes.

10. Stop: Add 50 µl of stop solution to each well. The color changes from blue to yellow.

11. Measure: Zero the microplate reader at 450 nm and measure the OD values within 15 minutes of adding the stop solution.

Precautions:

1. Let the kit equilibrate to room temperature for 15–30 minutes before use. Store unopened enzyme reagents in a sealed bag.

2. Concentrated washing solution may crystallize. Warm it in a water bath if needed; this does not affect results.

3. Use a pipette for accuracy. Control loading time within 5 minutes. Use a multichannel pipette for large numbers of samples.

4. Make a standard curve each time. If the sample OD is higher than the first standard, dilute the sample before testing. Multiply the result by the total dilution factor (×n×5).

5. Use a new sealing film for each experiment to avoid contamination.

6. Keep the substrate away from light.

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

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

9. Do not mix components from different batches.

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

Calculation:

Plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Draw a standard curve and determine the sample concentration based on its OD value. Alternatively, calculate the linear regression equation and substitute the sample’s OD value to find the actual concentration. Multiply by the dilution factor to get the final result.

(This image is for reference only)

Kit Performance:

1. The correlation coefficient (R) between the sample and expected concentration should be ≥0.95.

2. Intra-batch and inter-batch variation should be less than 9% and 11%, respectively.

Test Range:

40–800 µg/L

Storage Conditions and Expiration Date:

1. Storage: 2–8°C

2. Shelf life: 6 months

Xiamen Huijia Biotechnology Co., Ltd.

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