427275 Lactoferrin ELISA Kit

427275
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.



      The lactoferrin ELISA uses the sandwich enzyme-linked immunoassay method to detect lactoferrin in sample material.

      Catalogue Number427275
      Brand Family Calbiochem®
      Materials Required but Not Delivered Deionized water.
      Test tubes and beakers.
      Precision pipettes adjustable in the range of 0.1 to 1 ml.
      Automatic pipetters (100 and 50 µl) to add reagents into the plate wells.
      Incubator at 37° ± 1°C.
      Spectrophotometric microplate reader capable of measuring absorbance at 450 nm.
      References
      ReferencesAutiero, M., et al. 1991. J. Androl. 12, 191.
      Kähler, S., et al. 1988. Brit. J. Derm. 119, 289.
      Baynes, R.D., et al. 1986. Scand. J. Haematol. 36, 79.
      Birgens, H.S. 1985. Scand. J. Haematol. 34, 326.
      Lash, J.A., et al. 1983. Blood 61, 885.
      Bovaird, J.H., et al. 1982. Clin. Chem., 26, 2423.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, Lactoferrin Standard, Wash Buffer, Dilution Buffers, Anti-Lactoferrin Detector Antibody, Avidin-HRP, OPD Substrate, Stop Solution, and a user protocol.
      Applications
      Biological Information
      Assay time3 h
      Sample TypePlasma, urine, cell culture supernatant, bronchoalveolar lavage, and cerebrospinal fluid
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity>1 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/21/22-35-36/37/38-45-42/43

      Harmful by inhalation, in contact with skin and if swallowed.
      Causes severe burns.
      Irritating to eyes, respiratory system and skin.
      May cause cancer.
      May cause sensitization by inhalation and skin contact.
      S PhraseS: 30-26-36/37/39-45

      Never add water to this product.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, Lactoferrin Standard, Wash Buffer, Dilution Buffers, Anti-Lactoferrin Detector Antibody, Avidin-HRP, OPD Substrate, Stop Solution, and a user protocol.
      Specifications

      Documentation

      Lactoferrin ELISA Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Lactoferrin ELISA Kit Certificates of Analysis

      TitleLot Number
      427275

      References

      Reference overview
      Autiero, M., et al. 1991. J. Androl. 12, 191.
      Kähler, S., et al. 1988. Brit. J. Derm. 119, 289.
      Baynes, R.D., et al. 1986. Scand. J. Haematol. 36, 79.
      Birgens, H.S. 1985. Scand. J. Haematol. 34, 326.
      Lash, J.A., et al. 1983. Blood 61, 885.
      Bovaird, J.H., et al. 1982. Clin. Chem., 26, 2423.
      User Protocol

      Revision06-May-2009 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      BackgroundHuman lactoferrin (LTF) is an 80 kDa glycoprotein found in many mucosal secretions. Blood LTF is secreted from neutrophils and its plasma concentration is considered to be an indicator of neutrophil turnover. LTF's bactericidal properties are attributed to its strong iron binding capacity. During inflammation, LTF is often released into the extracellular medium from secondary granules of neutrophils. Therefore, its extracellular concentration can be used as an index of neutrophil activation, especially in blood samples containing anti-myeloperoxidase antibodies.
      Principles of the assayThe lactoferrin assay kit uses an enzyme-linked immunoassay (ELISA) method. Samples are incubated in the wells of a divided microplate that have been coated with a primary monoclonal antibody to LTF. The presence of LTF is detected using a biotinylated-monoclonal antibody to LTF. The final step in the assay uses amplification based on biotin-avidin coupling where avidin has been covalently linked to horseradish peroxidase (HRP). The amount of LTF is measured colorimetrically upon addition of the HRP substrate o-phenylenediamine (OPD).
      Materials provided• Sample Diluting Buffer (Kit Component No. KP1801): 3 bottles, 25 ml each, phosphate buffer pH 7.4, with NaCl, bovine serum albumin (BSA), Tween®-20 detergent, and 0.01% thimerosal. This solution is used to dilute biological samples
      • LTF Standard (Kit Component No. KP1802): 1 vial, 300 ng ± 5 ng of purified LTF, lyophilized.
      • Washing Buffer (20X) (Kit Component No. KP1803): 1 bottle, 100 ml, 1 M Tris-HCl buffer, pH 7.8, containing 3 M NaCl, 2% Tween®-20 detergent, and 0.01% merthiolate.
      • Anti-LTF Solution (Kit Component No. KP1804): 1 vial, 75 µl, Biotinylated monoclonal antibody to LTF in 20 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, 2 mg/ml BSA, 25% glycerol, and 0.01% merthiolate.
      • Avidin-HRP Solution (Kit Component No. KP1805): 1 vial, 75 µl, Avidin coupled to horseradish peroxidase in 50 mM Tris-HCl buffer, pH 8.0, containing 20 mg/ml BSA and 0.01% merthiolate.
      • Diluting Buffer (Kit Component No. KP1806): 3 vials, 8 ml each, 20 mM phosphate buffer, pH 7.4, with 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween®-20 detergent, and 0.01% merthiolate.
      • OPD Diluting Buffer (Kit Component No. KP1807): 1 bottle, 20 ml, 100 mM citrate buffer, pH 5.5, containing 0.012% H₂O₂, and 0.01 % merthiolate.
      • Stop Solution (Kit Component No. KP1808): 1 bottle, 20 ml, 1 M H2SO₄.
      • OPD Substrate (Kit Component No. KP1809): 4 tablets, each tablet contains 20 mg of OPD.
      • Microplates (Kit Component No. KP1810): One 96-well microplate supplied as 6x16-well strips, with frame.
      Materials Required but not provided Deionized water.
      Test tubes and beakers.
      Precision pipettes adjustable in the range of 0.1 to 1 ml.
      Automatic pipetters (100 and 50 µl) to add reagents into the plate wells.
      Incubator at 37° ± 1°C.
      Spectrophotometric microplate reader capable of measuring absorbance at 450 nm.
      Precautions and recommendations Do not smoke, eat or drink in areas where reagents and samples are handled.
      Do not pipette by mouth.
      Use pipettes with disposable tips to avoid bacterial contamination.
      Wear disposable gloves when handling reagents and samples.
      In case of accidental exposure of skin, eyes, or mucous membranes to Stop Solution, wash the exposed area thoroughly with water for 15 min.
      Human LTF was purified from human milk shown to be negative for HIV and HBV. LTF was heat-inactivated and treated by detergent in order to inactivate HIV. Handle LTF Standard, solution with the same precautions that are usually required for human blood products.
      PreparationNote: If necessary, dilute the samples with sample diluting buffer. Add 100 µl of sample per well. 1. Blood plasma Draw venous blood into a glass test tube that contains EDTA as an anticoagulant. Whole blood should be stored at 4°C. Centrifuge whole blood at 3000 x g for 10 min at 4°C and recover the resulting plasma supernatant. Plasma samples can be stored at 4°C for up to 24 h. For longer storage, freeze the samples at -20°C. Avoid freeze/thaw cycles. Ideally, plasma separation should be performed within 6 h. Plasma samples should be diluted 1:40 (v/v) in sample diluting buffer, for LTF values in the range 45-to-3000 ng/ml. With higher concentrations of LTF, greater dilutions will be required. Note: Heparin may interfere with LTF measurements. 2. Urine Urine should be recovered in clean flasks. Samples that are turbid, due to the presence of cellular or crystalline residues, should be clarified by centrifugation at 1000 x g for 10 minutes. Such samples can be stored at 4°C for up to 3 days, with or without pH adjustment. If the measured concentration of LTF is greater than 100 ng/ml in urine, dilute with sample diluting buffer. Otherwise, use undiluted sample. 3. Broncho-alveolar lavage (BAL) If the measured concentration of LTF is greater than 100 ng/ml, dilute with sample diluting buffer. Otherwise, use undiluted sample. 4. Cerebrospinal fluid If the measured concentration of LTF is greater than 100 ng/ml, dilute with sample diluting buffer. Otherwise, use undiluted sample. 5. Supernatants after centrifugation of cell cultures In a number of experimental conditions, LTF is released from human cells in culture. The Lactoferrin ELISA Kit can be used to assay LTF in culture medium that contains up to 20% fetal calf serum (FCS). However, if the measured concentration of LTF is greater than 100 ng/ml, samples should be diluted in sample diluting buffer
      Reagent preparationAll solutions must be kept at room temperature for 30 min before use. • Sample Diluting Buffer: The original solution is ready for use. • LTF Standard: Prepare a solution of LTF standard by adding 3.0 ml of Sample Diluting Buffer to the lyophilized protein. Exercise caution when opening the original vacuum-sealed bottle. The resulting solution may be stored at 4°C for up to 3 days. For longer term storage, aliquot and store at -70°C. To prepare a standard curve pipette 100 µl of sample diluting buffer in wells B1 through H1, as shown below. Well H1 will serve as the negative control. Pipette 200 µl of LTF standard, solution in well A1 as shown. Perform serial dilutions by transferring 100 µl from one well into the next one as shown. After each transfer, mix thoroughly using 4 suctions at each step with a pipette. Mix slowly in order to avoid foaming. Use a fresh pipette tip for each well. Cover the wells with adhesive paper and incubate at 37°C for 1 h.

      Figure 1: Organization of standards and samples on microplates.

      • Washing Solution (20x): The original buffer should be diluted 1:20 prior to use. Once prepared, the diluted washing buffer (1x) can be stored for up to 5 days at room temperature.

      Table 1: Washing solution dilutions

      • Biotinylated anti-LTF Solution: This reagent must be diluted ~1:250 in Diluting Buffer prior to use.

      Table 2: Anti-LTF solution dilutions

      • Avidin-HRP Solution: This reagent must be diluted ~1:250 in Diluting Buffer prior to use.

      Table 3: Avidin-HRP solution dilutions

      • OPD Solution: The OPD solution should be prepared 5 min prior to use. Dissolve the required number of OPD tablets in OPD Diluting Buffer. Tablets can be dissolved more quickly using a magnetic stirrer.

      Table 4: OPD solution dilutions

      Detailed protocol1. Organization of samples and standards on microplates

      Unpack the exact number of strips required for assay and set them on the frame. Determine the layout of standard and samples (see example below).

      Figure 2: Standard Curve Preparation


      2. Anti-LTF incubation

      Prepare the required volume of diluted biotinylated anti-LTF. Empty the microplate. Wash each well 5 times with washing buffer diluted 1:20. Drain the residual washing solution on absorbing paper. Add 100 µl of diluted biotinylated anti-LTF to each well. Cover the wells with adhesive paper and incubate at 37°C for 1 h.

      3. Avidin-HRP incubation

      Prepare the required volume of diluted avidin-horseradish peroxidase solution. Empty the microplate. Wash each well 5 times with washing buffer diluted 1:20. Drain the residual washing solution on absorbing paper. Add 100 µl of diluted avidin-horseradish peroxidase solution into each well. Cover the wells with adhesive paper and incubate at 37°C for 15 min.

      4. Colorimetric measurement
      Prepare the required volume of OPD solution. Empty the microplate. Wash each well 5 times with Washing Buffer diluted 1:20. Drain the residual washing solution on absorbing paper. Add 100 µl of prepared OPD solution to each well. Cover the wells with adhesive paper and incubate at 37°C for about 5-10 min until the absorbance of well number 1 (100 ng/ml) reaches approximately 1-1.5. Add 50 µl of Stop Solution into each well. Read the absorbance at 450 nm.
      CalculationsThe standard curve is obtained by plotting the absorbance at 450 nm as a function of the logarithm of standard LTF concentrations in ng/ml. Sample values are then obtained by interpolation or more accurately by using a third-order polynomial regression. The two modes of standard curve plotting are illustrated in Figure 3.

      Figure 3: Example of standard curves:

      (A) Third order polynomial regression (B) Interpolation.

      Sensitivity>1 ng/ml
      Sensitivity NotesFrom 36 repeated measurements performed on sample blank with the same microplate within a single day, the detection limit of the assay was found to be 1 ng/ml.
      Protocol SummaryQuick Reference Procedure for Lactoferrin ELISA Kit Material Preparation Protocol 1. Place solutions at room temperature 30 min before assay. 2. Prepare standard by adding 3 ml of diluting buffer to LTF standard. 3. Dilute wash buffer solution 1:20 with water. 4. Dilute anti-LTF 1:250 with diluting buffer. 5. Dilute avidin-alkaline phosphatase solution 1:250 with diluting buffer. 6. Dilute samples if necessary. 7. Prepare OPD solution 5 min before use. ELISA Protocol 1. Add 100 µl sample/standard to each well. 2. Cover plate and incubate for 1 h at 37°C. 3. Wash wells 5 times with washing buffer. 4. Add 100 µl diluted anti-LTF solution. 5. Cover plate and incubate for 1 h at 37°C. 6. Wash wells 5 times with washing buffer. 7. Add 100 µl diluted avidin-HRP solution. 8. Cover plate and incubate for 15 min at 37°C. 9. Wash wells 5 times with washing buffer. 10. Add 100 µl OPD solution. 11. Cover plate and incubate 5 to 10 min at 37°C. 12. Add 50 µl of stop solution. 13. Mix and read the absorbance at 450 nm. 14. Plot absorbance as a function of LTF concentration.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Tween® is a registered trademark of ICI Americas, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.