INTENDED USE

For the quantitative determination of activated   Lipopolysaccharides concentrations in serum, plasma, tissue homogenate, cell culture supernates and other biological fluids.

PRINCIPLE OF THE ASSAY

This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with one LPS  antibody. And then add sample, Biotinylated Ag. After incubation and washing, add Avidin-HRP, so as to form a coated Solid phase antibody-Biotinylated LPS -Avidin-HRP complex. The amount of bound labeled LPS  is inversely proportional to the amount of LPS  in the sample. TMB substrate solution is added to each well after wash the microtiter plate, TMB turn to blue catalyst with HRP. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of LPS  in the samples is then determined by comparing the OD of the samples to the standard curve.

SAMPLE COLLECTION AND STORAGES

Serum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.  Standard microplate reader capable of measuring absorbance at 450 nm

2.  Automated Washing

3.  37℃ incubator

4.  Clean tube and Eppendorf tube

5.  Distilled or deionized water

6.  Precision pipettes, disposable pipette tips, multi-channel pipettes and Absorbent paper

PRECAUTIONS

1.  The Stop Solution provided with this kit is an acid solution.

2.  Some components in this kit contain a preservative which may cause an allergic skin reaction. Avoid breathing mist.

3.  Chromogen Solution B may cause skin, eye, and respiratory irritation. Avoid breathing fumes.

4.  Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling.

WASHING METHOD

Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame. 

PROCEDURES IN SUMMARY

Prewarm all reagents to room temperature before assay.

Prepare reagents, samples in clean tubes or 96-well plate.

Transfer standard and samples to assay plate/strip, and add Biotinylated Ag and incubate 60 minutes at 37℃.

Plate-wash four times, add Avidin-HRP, incubate 30 minutes at 37℃.

Plate-wash four times, add Substrate A and B, incubate 15 minutes at 37℃ Add stop solution.

Measure within 10 minutes

ASSAY CHARACTERISTICS

1. Intra-assay CV (%) and Inter-assay CV (％) are less than 15%.

2. Assay range: 1 EU/L – 32 EU/L.

3. Sensitivity: The minimum detectable dose of LPS is typically less than 0.1 EU/L.

4. Cross-reactivity: This assay recognizes recombinant and natural  LPS. No significant cross-reactivity or interference was observed.

5. Storage: 2-8℃ .