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QIA112 Galectin-3 ELISA Kit

Galectin-3 ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
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QIA112
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.



      Designed for the quantitative detection of human Galectin-3.
      Catalogue NumberQIA112
      Brand Family Calbiochem®
      Application Data
      Recombinant, soluble, human galectin-3 was diluted in serial two-fold steps in Sample Diluent. The results represent the mean of three parallel titrations.
      Materials Required but Not Delivered• 5 ml and 10 ml Graduated pipettes
      • 10 µl to 1000 µl Adjustable single channel micropipettes with disposable tips
      • 50 µl to 300 µl Adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for the preparation of reagents
      • Multichannel wash bottle or automatic wash system for the delivery of wash solution
      • Plate reader capable of reading at 450 nm (optional reference wavelength 620 nm)
      • Distilled or deionized water
      • Statistical calculator with program to perform linear regression analysis
      References
      ReferencesLiu, F.T. 2000. In Lectins and Pathology. (Caron, M. and Seve, D. eds.) Harwood Academic Publishers, Amsterdam, The Netherlands.
      Sano, H., et al. 2000. J. Immunol. 165, 2156.
      Cooper, D.N., and Barondes, S.H. 1999. Glycobiology 9, 979.
      Kim, H.R., et al. 1999. Cancer Res. 59, 4148.
      Nachtigal, M., et al. 1998. Am. J. Pathol. 152, 1199.
      Bresalier, R.S., et al. 1997. Cancer 80, 776.
      Fernandez, P.L., et al. 1997. J. Pathol. 181, 80.
      Castronovo, V., et al. 1996. J. Pathol. 179, 43.
      Hsu, D.K., et al. 1996. Am. J. Pathol. 148, 1661.
      van den Brüle, F.A., et al. 1996. Hum. Pathol. 27, 1185.
      Yang, R.Y., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 6737.
      Dagher, S.F., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 1213.
      Liu, F.T., et al. 1995. Am. J. Pathol. 147, 1016.
      Sato, S., and Hughes, R.C. 1994. J. Biol. Chem. 269, 4424.
      van den Brüle, F.A., et al. 1994. Eur. J. Cancer 30A, 1096.
      Lindstedt, R., et al. 1993. J. Biol. Chem. 268, 11750.
      Liu, F.T. 1993. Immunol. Today 14, 486.
      Lotz, M.M., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 3466.
      Nangia-Makker, P., et al. 1993. Cancer Res. 53, 5033.
      Agrwal, N., et al. 1989. J. Biol. Chem. 264, 17236.
      Laing, J.G. and Wang, J.L. 1988. Biochemistry 27, 5329.
      Flotte, T.J., et al. 1983. Am. J. Pathol. 111, 112.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 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.
      Positive controlGalectin-3
      Applications
      Biological Information
      Assay range0.16 - 10 ng/ml
      Assay time4 h
      Sample TypeCell culture supernatants, plasma, serum, or other biological fluids
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity120 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe 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.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 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.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA112 0

      Documentation

      Galectin-3 ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA112

      References

      Reference overview
      Liu, F.T. 2000. In Lectins and Pathology. (Caron, M. and Seve, D. eds.) Harwood Academic Publishers, Amsterdam, The Netherlands.
      Sano, H., et al. 2000. J. Immunol. 165, 2156.
      Cooper, D.N., and Barondes, S.H. 1999. Glycobiology 9, 979.
      Kim, H.R., et al. 1999. Cancer Res. 59, 4148.
      Nachtigal, M., et al. 1998. Am. J. Pathol. 152, 1199.
      Bresalier, R.S., et al. 1997. Cancer 80, 776.
      Fernandez, P.L., et al. 1997. J. Pathol. 181, 80.
      Castronovo, V., et al. 1996. J. Pathol. 179, 43.
      Hsu, D.K., et al. 1996. Am. J. Pathol. 148, 1661.
      van den Brüle, F.A., et al. 1996. Hum. Pathol. 27, 1185.
      Yang, R.Y., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 6737.
      Dagher, S.F., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 1213.
      Liu, F.T., et al. 1995. Am. J. Pathol. 147, 1016.
      Sato, S., and Hughes, R.C. 1994. J. Biol. Chem. 269, 4424.
      van den Brüle, F.A., et al. 1994. Eur. J. Cancer 30A, 1096.
      Lindstedt, R., et al. 1993. J. Biol. Chem. 268, 11750.
      Liu, F.T. 1993. Immunol. Today 14, 486.
      Lotz, M.M., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 3466.
      Nangia-Makker, P., et al. 1993. Cancer Res. 53, 5033.
      Agrwal, N., et al. 1989. J. Biol. Chem. 264, 17236.
      Laing, J.G. and Wang, J.L. 1988. Biochemistry 27, 5329.
      Flotte, T.J., et al. 1983. Am. J. Pathol. 111, 112.
      User Protocol

      Revision14-August-2012 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe 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.
      BackgroundGalectin-3 is a 26 kDa β-galactoside-binding protein belonging to the Galectin family, which consists of more than ten members. It is composed of a carboxyl-terminal carbohydrate recognition domain (CRD) and amino-terminal tandem repeats. Galectin-3 is distributed in the epithelia of many organs and in various inflammatory cells, including macrophages, dendritic cells, and Kupfer cells. The expression of this lectin is up-regulated during inflammation, cell proliferation, cell differentiation, and through transactivation by viral proteins. Its expression is also affected by neoplastic transformation; up-regulation is found in certain types of lymphomas and thyroid carcinomas, while it is down-regulated in other types of malignancies in tissues such as colon, breast, ovarian, and uterine carcinomas. The expression of Galectin-3 shows a strong correlation with the grade and malignant potential of primary brain tumors. Increased Galectin-3 levels have also been noted in human atherosclerotic lesions. Galectin-3 has been shown to function through both intracellular and extracellular actions. It is a component of heterogeneous nuclear ribonuclear protein (hnRNP), a factor in pre-mRNA splicing and has been found to control cell cycle and to prevent T-cell apoptosis through interaction with Bcl-2 family members. This protein, which is secreted from monocytes/macrophages and epithelial cells has also been shown to function as an extracellular molecule in activating various types of cells such as monocytes/macrophages, mast cells, neutrophils, and lymphocytes. Galectin-3 has been shown to mediate cell-cell and cell-extracellular matrix interactions and acts as a novel chemoattractant for monocytes and macrophages.
      Principles of the assayThe Calbiochem® Galectin-3 ELISA Kit is a standard sandwich ELISA in which an anti-galectin-3 monoclonal antibody (specific for human galectin-3) is adsorbed onto the wells of a 96-well plate. Galectin-3 present in samples or standards binds to the antibody adsorbed to the plate. A biotin-conjugated anti-human galectin-3 detector antibody then binds to the galectin-3 captured by the first antibody. Unbound antibody is washed away and streptavidin-HRP is added and binds to the the biotin-conjugated ant-human galectin-3. Unbound Streptavidin-HRP is removed by another wash step, and a substrate solution reactive with HRP is added to the wells. A colored product is then formed in proportion to the amount of Galectin-3 present in the sample. The reaction is terminated by the addition of phosphoric acid and the absorbance is measured at 450 nm. A standard curve is then prepared from dilutions of a galectin-3 standard and the concentration of galectin-3 in the samples is determined.
      Materials provided• Coated 96-Well Plate (Kit Component No. JA7614): 1 plate, 96-wells, coated with mouse monoclonal antibody specific for human galectin-3
      • Biotin-Conjugated anti-human Galectin-3 polyclonal antibody (rabbit) (Kit Component No. JA7615): 1 vial, 100 µl
      • Streptavidin-HRP Concentrate (Kit Component No. JA7616): 1 vial, 150 µl
      • Galectin-3 Standard (Kit Component No. JA7603): 2 vials, lyophilized, 50 ng/ml upon reconstitution; please refer to the vial label for reconstitution volume
      • Assay Buffer Concentrate (Kit Component No. JA7604): 1 vial, 5 ml, supplied as 20X (PBS, 1% Tween® 20 detergent, 10% BSA)
      • Wash Buffer Concentrate (Kit Component No. JA7605): 1 bottle, 50 ml, supplied as 20X (PBS, 1% Tween® 20 detergent)
      • Sample Diluent (Kit Component No. JA7606): 1 bottle, 12 ml
      • Substrate Solution (Kit Component No. JA7617): 1 bottle, 15 ml, tetramethylbenzidine (TMB) and H₂O₂
      • Stop Solution (Kit Component No. JA7609): 1 bottle, 15 ml, 1 M phosphoric acid
      • Blue Dye (Kit Component No. JA7610): 1 vial, 400 µl
      • Red Dye (Kit Component No. JA7611): 1 vial, 400 µl
      • Green Dye (Kit Component No. JA7612): 1 vial, 400 µl
      • Plate Covers (Kit Component No. JA7613): 4 each
      Materials Required but not provided• 5 ml and 10 ml Graduated pipettes
      • 10 µl to 1000 µl Adjustable single channel micropipettes with disposable tips
      • 50 µl to 300 µl Adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for the preparation of reagents
      • Multichannel wash bottle or automatic wash system for the delivery of wash solution
      • Plate reader capable of reading at 450 nm (optional reference wavelength 620 nm)
      • Distilled or deionized water
      • Statistical calculator with program to perform linear regression analysis
      Precautions and recommendations1. Avoid contact of skin or mucous membranes with kit reagents or specimens.
      2. Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
      3. Use disposable pipette tips and/or pipettes to avoid microbial contamination or cross-contamination of reagents or specimens. Bacterial or fungal contamination of samples or reagents or cross-contamination between reagents may cause erroneous results.
      4. Bring substrate solutions to room temperature prior to use.
      5. As exact conditions vary from assay to assay, a standard curve must be established for every run.
      6. Use disposable pipette tips, flasks, and glassware; reusable glassware must be thoroughly washed and rinsed before use.
      7. Insufficient washing at any stage of the assay will result in either false positive or false negative results. Completely empty and fill wells with wash buffer as indicated for each wash cycle, do not allow wells to sit uncovered or dry for extended periods.
      8. Do not mix or substitute reagents with those form other lost or products from other sources.
      9. Do not expose kit reagents to strong light during storage or incubation.
      10. Do not pipette by mouth.
      11. Do not eat or smoke in areas where kit reagents or samples are handled.
      12. Avoid contact of substrate solution with oxidizing agents or metal.
      13. Avoid splashing or generation of aerosols.
      14. Use clean, dedicated trays for dispensing the conjugate and substrate reagents.
      15. Exposure to acids wil inactivate the conjugate.
      16. Glass-distilled water or deionized water must be used for reagent preparation.
      17. Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1% sodium hypochlorite. Allow 30 min for effective decontamination. Liquid waste containing acid must be neutralized prior to adding sodium hypochlorite.
      PreparationNote: 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.
      Reagent preparationWash 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.
      Detailed protocol1. Remove the desired number of strips from the Coated 96-Well Plate, return the unused strips to the foil pouch, and store at 4°C. Each sample, standard, blank, and
      control should be assayed at least in duplicate.
      2. Wash the strips twice, using approximately 300 µl Wash Buffer per well. Thoroughly aspirate the contents between washes. Do not scratch the inner surface of the microwells. Following the final wash, empty the wells completely and tap on an absorbent pad or paper towel to remove excess buffer. Use the strips immediately after washing or place upside down on a wet absorbent paper for up to 15 min. Do not allow wells to dry.
      3. In a separate polypropylene 96-well plate or a set of polypropylene tubes (do not use polystyrene), prepare a series of Galectin-3 Standard dilutions (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0 ng/ml) with Sample Diluent. Allow two wells/tubes for each dilution and slightly more than 100 µl for each dilution. Transfer 100 µl of each Galectin-3 Standard to wells that correspond to the first two columns/strips of the ELISA plate (i.e. wells A1 through H1 and A2 through H2). Start with 0 ng/ml at the bottom of the strip (e.g., wells H1 and H2) and move up to the 10 ng/ml dilution at the top of the strips (e.g., wells A1 and A2).
      4. Add 50 µl Sample Diluent to designated sample wells. If samples are too concentrated they can be diluted with the sample diluent before addition to the wells. Add 50 µl Sample Diluent to designated Blank wells (in duplicate).
      5. Add 50 µl of each sample, in duplicate, to the designated wells.
      6. Add 50 µl diluted Biotin-Conjugated anti-human Galectin-3 polyclonal antibody to all wells.
      7. Cover with a Plate Cover and incubate at room temperature for 2 h on a rotator set at 200 rpm.
      8. Remove Plate Cover and empty wells. Wash the wells 3 times as outlined in step 2.
      9. Add 100 µl diluted Streptavidin-HRP to all wells.
      10. Cover with a Plate Cover and incubate at room temperature for 1 h on a rotator set at 200 rpm.
      11. Remove the Plate Cover and empty wells. Wash the wells 3 times as in step 2.
      12. Add 100 µl TMB Substrate Solution to all wells.
      13. Incubate at room temperature for approximately 10 min. Avoid direct exposure to light. Please note that the point at which the substrate reaction should be stopped may be determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 absorbance points, therefore the color development must be watched carefully at this point in the assay. The substrate reaction must be stopped before the positive wells are no longer properly recordable.
      14. Stop the enzyme reaction by quickly adding 100 µl Stop Solution to all wells. It is important that the Stop Solution be spread quickly and uniformly throughout the wells to completely inactivate the enzyme. The results must be read immediately after the Stop Solution is added or within 1 h if the strips are stored at 4°C in the dark.
      15. Read the absorbance using a 96-well plate reader set at 450 nm as the primary wavelength and (optional) 620 nm as the reference wavelength (610 nm to 650 nm is acceptable). Blank the plate reader using the blank wells. Determine the absorbance of both the samples and the Galectin-3 standards. Incubation without shaking may result in absorbance values lower than shown below. These results are still valid.
      Calculations1. 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.
      Assay characteristics and examplesPanels of 16 human serum samples and 10 human plasma samples were assayed for levels of human galectin-3 as outlined in the Detailed Protocol. The level of galectin-3 detected in serum samples ranged from 620 pg/ml to 6.25 ng/ml with a mean level of 2.33 ng/ml. The level of galectin-3 detected in plasma samples ranged from 4.67 ng/ml to 10.3 ng/ml with a mean level of 7.07 ng/ml.
      Standard curve

      Figure 1: Standard Curve

      Recombinant, soluble, human galectin-3 was diluted in serial two-fold steps in Sample Diluent. The results represent the mean of three parallel titrations.






      Table 1: Typical Data Using the Galectin-3 Standard
      Sensitivity120 pg/ml
      Sensitivity NotesThe limit of detection of Galectin-3 defined as the analyte concentration resulting in an absorption value significantly higher than that of the dilution medium was determined to be <120 pg/ml (mean of 6 independent assays).
      Assay Range0.16 - 10 ng/ml
      RecoveryThe spike recovery was evaluated by spiking four levels of Galectin-3 into pooled human serum. Recoveries were determined in three independent experiments with 6 replicates each. The unspiked diluent was used as a blank in these experiments. Recoveries ranged from 73% to 88% with an overall mean recovery of 76.7%.
      ReproducibilityIntra-Assay Reproducibility within the assay was evaluated in three independent experiments. Each assay was carried out with 6 replicates of 6 serum samples containing different concentrations of human galectin-3. Two standard curves were run on each plate. Data below show the mean human galectin-3 concentration and the coefficient of variation for each sample. The overall intra-assay coefficient of variation was determined to be 6.4%





      Table 2: Intra-Assay Reproducibility

      Inter-Assay Assay to assay reproducibility within one laboratory was evaluated in three independent experiments carried out by three technicians. Each assay was carried out with 6 replicates of 6 serum samples containing different concentrations of human galectin-3. Two standard curves were run on each plate. The data below show the mean human galectin-3 concentration and the coefficient of variation calculated on 18 determinations of each sample. The overall coefficient of variation has been calculated to be 11.4%.

      Table 3: Inter-Assay Reproducibility
      ParallelismThree serum samples with different levels of human galectin-3 were analyzed at four serial two-fold dilutions (1:2–1:16) with 4 replicates each. The percent recovery of expected values is listed in the table below. Recoveries ranged from 94% to 128% with an overal mean recovery of 115.3%.

      Table 4: Parallelism
      SpecificityGalectin-3 positive serum spiked with physiologically relevant concentrations of circulating factors of the immune system showed no detectable cross reactivity.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
      Tween® is a registered trademark of ICI Americas, Inc.