Galectin-3 ELISA Kit

Galectin-3 positive serum spiked with physiologically relevant concentrations of circulating factors of the immune system showed no detectable cross reactivity.

The Calbiochem Galectin-3 ELISA Kit is designed for the quantitative detection of human Galectin-3 in cell culture supernatants and biological samples such as serum and plasma.

Assay Time: 4 h

Wash Buffer: If crystals are observed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. To prepare enough Wash Buffer for the entire plate, add 50 ml Wash Buffer Concentrate to 950 ml distilled or deionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Store the Wash Buffer at 4°C or room temperature; Wash Buffer is stable for up to 30 days.Assay Buffer: To prepare enough Assay Buffer for the entire plate, add 5 ml Assay Buffer Concentrate to 95 ml distilled or deionized water. Mix gently to avoid foaming. Store at 4°C. Assay Buffer is stable for up to 30 days.Biotin conjugate Anti-human Galectin-3 polyclonal antibody: Prior to diluting the Biotin conjugate Anti-human Galectin-3 polyclonal antibody, add 60 µl Green-dye to 6 ml Assay Buffer. Dilute the Biotin conjugate Anti-human Galectin-3 polyclonal antibody 1:100 with Assay Buffer containing Green Dye. To prepare enough Biotin conjugate Anti-human Galectin-3 polyclonal antibody for the entire plate, add 60 µl Biotin conjugate Anti-human Galectin-3 polyclonal antibody to 5.94 ml Assay Buffer.Galectin-3 Standard: Reconstitute the Galectin-3 Standard by adding the volume of distilled water as stated on the vial label. Mix gently to ensure solubilization. Store reconstituted standard promptly at -20°C. Discard after one week.Streptavidin-HRP: Prior to diluting the Streptavidin-HRP Concentrate, add 48 µl Red Dye to 12 ml Assay Buffer. Dilute the Streptavidin-HRP Concentrate 1:400 in Assay Buffer containing Red Dye. To prepare enough Streptavidin HRP for the entire plate, add 30 µl Streptavidin-HRP Concentrate to 11.970 ml Assay Buffer containing Red Dye. Store stock solution at 4°C.TMB Substrate Solution Warm Substrate Solution to room temperature before use. While these TMB substrate solution may develop a yellow tinge over time, it does not seem to affect product performance. A blue color present in the TMB substrate solution indicates that they have been contaminated and must be discarded. Avoid direct exposure of TMB reagents to intense light and oxidizing agents during storage or incubation.

Note: Remove serum from clots or red cells, respectively, as soon as possible after clotting and separation. Samples containing visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Clinical samples should be kept at 4°C and separated quickly before storing at -20°C to avoid the loss of bioactive galectin-3. Samples to be assayed within 24 h may be stored at 4°C. Avoid repeated freeze-thaw cycles. To avoid mistakes in pipetting, dilute the Blue-dye 1:250 in the PBS that will be used to dilute samples (optional).Note on Stability of Samples: Aliquots of serum samples (unspiked and spiked with galectin-3) were stored at -20°C, thawed up to 5 times, and the level of human galectin-3 determined using the Galectin-3 ELISA Kit. There was no significant loss of galectin-3 immunoreactivity up to 5 freeze/thaw cycles. Aliquots of a serum sample (unspiked and spiked with galectin-3) were also stored at -20°C, 4°C, room temperature, and 37°C and the level of human galectin-3 was determined using the Galectin-3 ELISA Kit. There was no significant loss of galectin-3 immunoreactivity during storage under these conditions.

Upon arrival store the entire contents of the kit at 4°C.

1. Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 percent of the mean.2. Create a standard curve by plotting the mean absorbance for each standard concentration on the y-axis against the galectin-3 concentration on the x-axis. Draw a best fit curve through the points on the graph.3. To determine the concentration of the Galectin-3 in each sample find the mean absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding Galectin-3 concentration. If samples have been diluted the concentration read from the standard curve must be multiplied by the respective dilution factor. The evaluation of samples with an absorbance exceeding 2.0 may result in incorrect/low Galectin-3 levels. These samples should be re-analyzed at a higher dilution to precisely quantitate the Galectin-3 level. It is recommended that each laboratory establish control samples of known Galectin-3 concentration to run with each assay. If the values obtained are not within the expected range of these controls, the assay results may be invalid.

Positive Control
Galectin-3

Coated Microplate, Anti-Galectin-3 (Rabbit), Anti-Rabbit-HRP, Galectin-3 Standard (2 vials), Assay Buffer Concentrate, Wash Buffer Concentrate, Sample Diluent, Substrate Solution I, Substrate Solution II, Stop Solution, Blue-Dye, Red-Dye, Green-Dye, Plate Covers, and a user protocol.

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CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Toxicity: Multiple Toxicity Values, refer to MSDS (O)