Ferritin ELISA Kit

Catalog No. ABIN649047

Product Details Ferritin ELISA Kit

Target: Ferritin (FE)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Immunoenzymometric sequential assay (TYPE 4): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti- ferritin antibody. Upon mixing monoclonal biotinylated antibody, and a serum containing the native antigen, reaction results between the native antigen and the antibody, forming an antibody-antigen complex. Simultaneously the biotin attached to the antibody binds to the streptavidin coated on the microwells resulting in immobilization of the complex. After a suitable incubation period, the antibody-antigen bound fraction is separated from unbound antigen by decantation or aspiration. Another antibody (directed at a different epitope) labeled with an enzyme is added. Another interaction occurs to form an enzyme labeled antibody-antigen-biotinylated-antibody complex on the surface of the wells. Excess enzyme is washed off via a wash step. A suitable substrate is added to produce color measurable with the use of a microplate spectrophotometer. The enzyme activity on the well is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. Ferritin Calibrators (1ml / vial). Six vials of Ferritin calibrators at levels of 0 (A), 10 (B), 50 (C), 150 (D), 400 (E) and 800 (F) ng/ml. Store at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 3rd IS 94/572. B. Ferritin Biotin Reagent (13ml/vial). One vial containing biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. Ferritin Enzyme Reagent (13 ml/vial). One vial containing Horseradish Peroxidase (HRP) labeled anti-ferritin IgG in buffer, dye and preservatives. Store at 2-8°C. D. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. E. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate A (7ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. G. Substrate B (7ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8m/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. I. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Material not included: 1. Pipette capable of delivering 25, & 50µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Quality control materials.

Application Details

Application Notes: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA licensed reagents. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Sample Volume: 25 μL

Plate: Pre-coated

Reagent Preparation:

1. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature (20-27°C) for up to 60 days. 2. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Sample Collection: The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants. Allow the blood to clot for samples. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 050ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of ferritin in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding ferritin concentration in ng/ml on linear graph paper. 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of ferritin for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). Note: Computer data reduction software designed for IEMA. Elisa assays may also be used for the data reducton.

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 025 ml (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of the Ferritin Biotin Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 30 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of the Ferritin Enzyme Conjugate to each well. DO NOT SHAKE THE PLATE AFTER ENZYME ADDITION. 9. Incubate 30 minutes at room temperature. 10. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 11. Add 300µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. 12. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 13. Incubate at room temperature for 15 minutes. 14. Add 0. 050ml (50µl) of stop solution to each well and mix gently for 15-20 seconds. 15. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution. Note: Always add reagents in the same order to minimize reaction time differences between wells.

Storage: 4 °C/-20 °C

Target Details for Ferritin

Target: Ferritin (FE)

Alternative Name: Ferritin (FE Products)

Synonyms: 1HCH ELISA Kit, BEST:LD36673 ELISA Kit, CG2216 ELISA Kit, Dmel\\CG2216 ELISA Kit, FER1 ELISA Kit, FER1HCH ELISA Kit, Fer-L ELISA Kit, Fer1 ELISA Kit, Fer1HC ELISA Kit, FerHCH ELISA Kit, HCH ELISA Kit, T21 ELISA Kit, T9 ELISA Kit, fer1 ELISA Kit, fer1hch ELISA Kit, l(3)00451 ELISA Kit, l(3)j10B4 ELISA Kit, SFerH-1 ELISA Kit, SOF-35 ELISA Kit, SOF-5L ELISA Kit, NV13011 ELISA Kit, ACP1 ELISA Kit, F8A5.24 ELISA Kit, F8A5_24 ELISA Kit, Ferritin 1 heavy chain homologue ELISA Kit, ferritin light chain ELISA Kit, ferritin ELISA Kit, chromosome 26 open reading frame, human C10orf131 ELISA Kit, hyaluronan and proteoglycan link protein 2 ELISA Kit, NAD(P)-linked oxidoreductase superfamily protein ELISA Kit, Fer1HCH ELISA Kit, H-1 ELISA Kit, YPDSF_1340 ELISA Kit, ftnA ELISA Kit, Ft ELISA Kit, C26H10orf131 ELISA Kit, HAPLN2 ELISA Kit, AT1G60730 ELISA Kit

Background: Summary and Explanation of the test: Ferritin, in circulation, as measured in serum levels is a satisfactory index of body's iron storage. The iron storage is directly measured by quantitative phlebotomy, iron absorption studies, liver biopsies and microscopic examinations of bone marrow aspirates. Iron deficiency (Anemia) and iron overload (Hemochromatosis) are conditions associated with body's iron storage or lack thereof. Measurements of total iron binding capacity (TIBC) have widely been used as aids in the determination of these conditions. However, an assay of serum Ferritin is simply more sensitive and reliable means of demonstration these disorders. Ferritin is present in blood in very low concentrations. Normally, approximately 1% of plasma iron is contained in Ferritin. The plasma ferritin, is in equilibrium with body stores, and variations of iron storage. The plasma concentrations of ferritin decline very early in anemic conditions like development of iron deficiency, long before the changes are observed in the blood hemoglobin concentration, size of the erythrocytes and TIBC. Thus measurements of serum ferritin can serve as an early indicator of iron deficiency that is uncomplicated by other concurrent conditions. At the same time a large number of chronic conditions can result in elevated levels of serum ferritin. These include chronic infections, chronic inflammatory diseases such as rheumatoid arthritis, heart disease and some other malignancies, especially lymphomas, leukemia, breast cancer and neuroblastoma. In patients who have these chronic disorders together with iron deficiency, serum ferritin levels are often normal. An increase in circulating ferritin is observed in patients with viral hepatitis or after a toxic liver injury as a release of ferritin from the injured liver cells. Elevated serum ferritin levels are found in patients with hemochromatosis and hemosiderosis. Circulating ferritin levels have been used by clinicians, as an aid, in the diagnosis of several other disorders. It has proved as a valuable tool in differential diagnosis of anemia due to iron deficiency and anemias due to other disorders and, in exposing the depletion of iron reserves long before the onset of anemia. Serial determinations have been used to monitor, non-invasively, the erosion of iron storage during pregnancy and in patients undergoing dialysis. Serum ferritin is routinely used as a screen for iron deficiency for a variety of populations like blood donors and people who are receiving regular blood transfusions or iron replacement therapy. In this method, ferritin calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal antibody (specific for ferritin) is added and the reactants mixed. Reaction results between the biotinylated ferritin antibody and native ferritin to form an immune complex that is deposited on the streptavidin coated well. The excess serum proteins are washed away via a wash step. Another ferritin specific antibody, labeled with an enzyme, is added to the wells. The enzyme labeled antibody binds to the ferritin already immobilized on the well. Excess enzyme is washed off via a wash step. A color is generated by the addition of a substrate. The intensity of the color generation is directly proportional to the concentration of the ferritin in the sample. The employment of several serum references of known ferritin levels permits the construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with ferritin concentration. Intended Use: The Quantitative Determination of Circulating Ferritin Concentrations in Human Serum by a Microplate Immunoenzymometric assay.

Pathways: Transition Metal Ion Homeostasis