Lactoferrin ELISA Kit

Catalog No. ABIN612725

Product Details Lactoferrin ELISA Kit

Target: Lactoferrin (LTF) (Lactotransferrin (LTF))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 0.1 ng/mL

Application: ELISA

Purpose: The AssayMax Human Lactoferrin ELISA kit is designed for detection of human plasma, serum, urine, salvia, milk and other body fluids

Brand: AssayMax

Sample Type: Plasma

Analytical Method: Quantitative

Specificity: If cell culture supernatants contains, FBS, the minimum detectable dose of human lactoferrin will be 1 ng/ml.

Components: Human Lactoferrin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human lactoferrin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Lactoferrin Standard: Human Lactoferrin in a buffered protein base (800 ng, lyophilized). Biotinylated Lactoferrin Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against human lactoferrin (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water

Application Details

Sample Volume: 50 μL

Assay Time: < 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique, which measures lactoferrin in less than 4 hours. A polyclonal antibody specific for lactoferrin has been pre-coated onto a microplate. Lactoferrin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for lactoferrin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 800 ng of Lactoferrin Standard with 5 ml of MIx Diluent to generate a stock solution of 160 ng/ml. Allow the stock solution to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare the standard solution by diluting the stock solution (160 ng/ml) 1:4 with MIx Diluent to produce 40ng/ml. Prepare triplicate points by serially diluting the standard (40ng/ml) 1:2 using equal volume of MIx Diluent to give 20, 10, 5, 2.5, 1.25, and 0.625 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:200 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:200 into MIx Diluent. Store serum at -20°C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:10 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva to sample tube. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:1000 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 2 Milk: Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:100000 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles.

Assay Procedure:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated Lactoferrin Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer as above. 3 Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for about 8 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results:

Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.1% and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: The kit should not be used beyond the expiration date.

Storage: 4 °C/-20 °C

Storage Comment: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.

References for Lactoferrin ELISA Kit (ABIN612725)

Mayeur, Veilleux, Pouliot, Lamarche, Beaulieu, Hould, Richard, Tchernof, Levy: "Plasma Lactoferrin Levels Positively Correlate with Insulin Resistance despite an Inverse Association with Total Adiposity in Lean and Severely Obese Patients." in: PLoS ONE, Vol. 11, Issue 11, pp. e0166138, (2016) (PubMed).

Woods, Tan, Ullah, Frauenfelder, Ooi, Carney: "The effect of nasal irrigation formulation on the antimicrobial activity of nasal secretions." in: International forum of allergy & rhinology, (2015) (PubMed).

Jones, Thatcher, March, Davison: "Influence of 4 weeks of bovine colostrum supplementation on neutrophil and mucosal immune responses to prolonged cycling." in: Scandinavian journal of medicine & science in sports, (2015) (PubMed).

Glennie, Gritzfeld, Pennington, Garner-Jones, Coombes, Hopkins, Vadesilho, Miyaji, Wang, Wright, Collins, Gordon, Ferreira: "Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage." in: Mucosal immunology, (2015) (PubMed).

Shiga, Oshima, Kojima, Sugimoto, Tamaki, Murata, Takeuchi, Sato: "Recombinant human lactoferrin-Fc fusion with an improved plasma half-life." in: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, Vol. 67, pp. 136-43, (2014) (PubMed).

Tiwari, Ali, Balla, Naik, Honavar, Reddy, Vemuganti: "Establishing human lacrimal gland cultures with secretory function." in: PLoS ONE, Vol. 7, Issue 1, pp. e29458, (2012) (PubMed).

Zagierski, Szlagatys-Sidorkiewicz, Jankowska, Krzykowski, Korzon, Kaminska: "Maternal smoking decreases antioxidative status of human breast milk." in: Journal of perinatology : official journal of the California Perinatal Association, Vol. 32, Issue 8, pp. 593-7, (2012) (PubMed).

Target Details for Lactoferrin

Target: Lactoferrin (LTF) (Lactotransferrin (LTF))

Alternative Name: Lactoferrin (LTF Products)

Synonyms: LTF ELISA Kit, GIG12 ELISA Kit, HLF2 ELISA Kit, LF ELISA Kit, Lf ELISA Kit, Csp82 ELISA Kit, MMS10R ELISA Kit, Ms10r ELISA Kit, PLF ELISA Kit, TFEW ELISA Kit, conalbumin ELISA Kit, lactotransferrin ELISA Kit, transferrin ELISA Kit, transferrin (ovotransferrin) ELISA Kit, LTF ELISA Kit, CpipJ_CPIJ000114 ELISA Kit, TF ELISA Kit, Ltf ELISA Kit

Background: Lactoferrin is an 80 kDa iron-binding glycoprotein produced by many exocrine glands with a major constituent in the secondary granules of neutroplilic leukocytes. Serum lactoferrin concentration is much higher during inflammation. Lactoferrin is known to be an immune modulator or enhancer due to specific receptors for lactoferrin that are found on many key immune cells such as lymphocytes, monocytes and macrophages, and is known to be directly involved in the up-regulation of natural killer (NK) cell activity. Lactoferrin is present in maternal milk, saliva, tears, vaginal secretions, semen, bronchoalveolar lavage fluid, and specific granules of polymorphonuclear leukocytes (PMNs). Lactoferrin is found mainly in the oral cavity where it can come into direct contact with pathogens such as viruses, bacteria, etc. Lactoferrin directly inhibits viruses by binding to viral receptor sites, thus preventing the virus from infecting healthy cells. Lactoferrin has a direct bacteriacidal function to certain bacteria such as Streptococcus mutans, Vibrio cholerae, Escherichia coli, Actinobacillus actinomycetemcomitans, and Legionella pneumophila. Also, it has a bacteriostatic effect that deprives iron-requiring bacteria of this essential growth nutrient. Lactoferrin is also considered an antioxidant that scavenges free iron, helping to prevent uncontrolled iron based free radical reactions, thus protecting certain cells from peroxidation.

Pathways: Transition Metal Ion Homeostasis