INTENDED USE

For the quantitative determination of activated   D-Lactic acid 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 D-LA  antibody. And then add sample, Biotinylated Ag. After incubation and washing, add Avidin-HRP, so as to form a coated Solid phase antibody-Biotinylated D-LA -Avidin-HRP complex. The amount of bound labeled D-LA  is inversely proportional to the amount of D-LA  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 D-LA  in the samples is then determined by comparing the OD of the samples to the standard curve.

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. 

ASSAY PROCEDURE

1. First, secure the desired number of coated wells in the holder. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

2. Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.

3. Add 50μl of Biotinylated Ag to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

4.  Wash the Microtiter Plate 4 times.(See WASHING METHOD)

5. Add 50μl of Avidin-HRP to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 30 minutes at 37°C.

6.  Wash the Microtiter Plate 4 times.(See WASHING METHOD)  

7. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

8. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

9.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

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: 1.5 μmol/L – 48 μmol/L.

3. Sensitivity: The minimum detectable dose of D-LA is typically less than 0.1 μmol/L.

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

5. Storage: 2-8℃ .