Haptoglobin ELISA Kit

Produktnummer ABIN956036

Dieses Colorimetric ELISA kit dient der quantitativen Messung von Schwein Haptoglobin.

Kurzübersicht für Haptoglobin ELISA Kit (ABIN956036)

Target: Haptoglobin (HP)

Reaktivität: Schwein

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Produktdetails Haptoglobin ELISA Kit

Verwendungszweck: The Pig Haptoglobin ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of Haptoglobin in pig biological fluids.

Analytische Methode: Quantitative

Produktmerkmale: In this assay the haptoglobin present in the sample reacts with the anti-Hp antibodies, which have been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound sample proteins by washing, anti-Hp antibodies conjugated with horseradish peroxidase (HRP), are added. This HRP-conjugated antibody forms a complex with the previously bound haptoglobin. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of haptoglobin in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of haptoglobin in the test sample. The quantity of haptoglobin in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected for sample dilution.

Bestandteile: 1. Diluent Concentrate: One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate: One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate: One vial containing 150 µL of a 100X concentrated affinity-purified anti-pig haptoglobin antibody conjugated with HRP in a stabilizing buffer.
4. TMB Substrate Solution: One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution: One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate: Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-pig haptoglobin.
7. Pig Haptoglobin Calibrator: One vial containing a lyophilized Pig Haptoglobin Calibrator.
8. Positive Control: One vial containing 50 µL of serum with 0.1% sodium azide. See the Control Certificate for the concentration.

Benötigtes Material: Test tubes
Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Microplate washer/aspirator
Distilled or de-ionized H2O Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer
Vortex mixer

Anwendungsinformationen

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Diluent Concentrate: The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
   2. Wash Solution Concentrate: The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to 30-35°C before dilution can dissolve crystals.
   3. Enzyme-Antibody Conjugate Concentrate: Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoid foaming.
   4. TMB Substrate Solution: Ready to use as supplied.
   5. Stop Solution: Ready to use as supplied.
   6. Microtiter Plate: Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will not be used in the assay and place back in pouch and re-seal along with desiccant.
   7. Pig Haptoglobin Calibrator: Add 2.0 mL of distilled or de-ionized water to the lyophilized Pig Haptoglobin Calibrator and mix gently until dissolved. The calibrator is now at a concentration of 56.8 µg/mL (the reconstituted calibrator should be aliquoted and frozen if future use is intended). Prepare the Pig Haptoglobin Calibrators immediately prior to use according to the table below. Mix well between each step. Avoid foaming.
   8. Positive Control: The concentration and recommended dilution are provided on the Control Certificate. Before use, briefly centrifuge the Positive Control to allow all of the liquid to collect in the bottom of the vial.

Ergebnisberechnung:

1. Subtract the average background value from the test values for each sample.
   2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a four- parameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
   3. Interpolate test sample values from the calibration curve. Correct for sample dilution factor to arrive at haptoglobin concentration in original sample.
   
   1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the information contained in the package insert instructions and with adherence to good laboratory practice.
   2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware, quality of distilled or de-ionized water, and accuracy of reagent and sample pipetting, washing technique, incubation time or temperature.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung: 1. Complete Kit: The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: See long-term storage recommendations below for the Pig Haptoglobin Calibrator and Positive Control.
2. Diluent: The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution: The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate: Undiluted anti-haptoglobin-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The working conjugate solution is stable for up to 8 hours.
5. TMB Substrate Solution: The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
6. Stop Solution: The Stop Solution should be stored at 4°C and is stable until the expiration date.
7. Microtiter Plate: Anti-pig haptoglobin coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouch with a desiccant pack.
8. Pig Haptoglobin Calibrator: The lyophilized Pig Haptoglobin Calibrator should be stored at 4°C or frozen until reconstituted. The reconstituted calibrator should be aliquoted and stored frozen. Avoid multiple freeze/thaw cycles. The working calibrator solutions should be prepared immediately prior to use and are stable for up to 8 hours.
9. Positive Control: For storage longer than 7 days keep frozen until the expiration date. Storage less than 7 days can be at 4°C. Avoid multiple freeze/thaw cycles.

Haltbarkeit: The expiry date is stated on the label.

Details zu Haptoglobin

Target: Haptoglobin (HP)

Andere Bezeichnung: Haptoglobin

Hintergrund: Acute phase proteins are plasma proteins, which increase in concentration following infection, inflammation or trauma. The first acute phase protein to be recognized was discovered in humans by Tillet and Frances in 1930. Haptoglobin (Hp) is a heterogeneous plasma protein mostly synthesized by the liver. The haptoglobin monomer consists of two heavy chains, beta chains (40 kDa) and two light chains, alpha chains, alpha 1 (9 kDa) and alpha 2 (16 kDa) that are linked by disulfide bonds. The three major haptoglobin types are, Hp1-1, which is monomeric (98 kDa), Hp1-2 is polymeric at about 200 kDa, and Hp2-2 at about 400 kDa. The haptoglobin level in serum rises quickly following acute tissue damage within 24 to 48 hours and also falls very rapidly once the stimulus is removed. In fact, haptoglobin levels are decreased in hemolytic anemia. Haptoglobin has a high affinity for hemoglobin (Hb) and its function appears to be to prevent loss of hemoglobin in urine, which would lead to loss of iron. Investigations over the past few years have shown that quantification of haptoglobin in plasma or serum can provide valuable information in the detection, prognosis, and monitoring of disease not only in humans, but in companion animals and farm herds as well.

Pathways: Transition Metal Ion Homeostasis