IgA ELISA Kit

Produktnummer ABIN956197

Hund IgA ELISA Kit, Colorimetric Assay für die Quantifizierung von Hund IgA.

Kurzübersicht für IgA ELISA Kit (ABIN956197)

Target: IgA

Reaktivität: Hund

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Produktdetails IgA ELISA Kit

Verwendungszweck: The Dog IgA ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of IgA in dog biological samples.

Analytische Methode: Quantitative

Produktmerkmale: In this assay the IgA present in samples reacts with the anti-IgA antibodies which have been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-IgA antibodies conjugated with horseradish peroxidase (HRP), are added. These enzyme-labeled antibodies form complexes with the previously bound IgA. 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 varies directly with the concentration of IgA in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of IgA in the test sample. The quantity of IgA 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-dog IgA antibody conjugated with HRP in 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-dog IgA.
7. Dog IgA Calibrator: One vial containing Dog IgA Calibrator.

Benötigtes Material: Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Test tubes
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. Dog IgA Calibrator: The calibrator is now at a concentration of 1.735 mg/mL. Dog IgA Calibrators need to be prepared immediately prior to use. Mix well between each step. Avoid foaming.

Testdurchführung:

Dilution of Samples Due to the high sensitive nature of the assay each sample should be diluted before use for a normal assay. A 1:4,000 dilution of serum or plasma is appropriate for most samples. For absolute quantification of samples that yield results outside the range of the calibration curve, a lesser or greater dilution might be required. To prepare a 1:4,000 dilution of sample, transfer 2 µL of sample to 198 µL of 1X Diluent. This gives you a 1:100 dilution. Mix thoroughly. Next, dilute the 1:100 samples by transferring 20 µL to 780 µL of 1X Diluent. You now have a 1:4,000 dilution of your sample. Mix thoroughly at each stage. Procedure Bring all reagents to RT before use. 1. Pipette 100 µL of Calibrator 0 (0.0 ng/mL) in duplicate Calibrator 1 (31.25 ng/mL) in duplicate Calibrator 2 (62.5 ng/mL) in duplicate Calibrator 3 (125 ng/mL) in duplicate Calibrator 4 (250 ng/mL) in duplicate Calibrator 5 (500 ng/mL) in duplicate Calibrator 6 (1,000 ng/mL) in duplicate
2. Pipette 100 µL of diluted sample (in duplicate) into pre-designated wells.
3. Incubate the Microtiter Plate at 22°C (RT) for thirty (30 ﾱ 2) minutes. Keep plate covered and level during incubation.
4. Following incubation, aspirate the contents of the wells.
5. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of four washes. If washing manually: completely fill wells with diluted Wash Solution, invert the plate and pour/shake out the contents in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual Wash Solution. Repeat three times for a total of four washes.
6. Pipette 100 µL of appropriately diluted Enzyme-Antibody Conjugate to each well. Incubate at 22°C (RT) for thirty (30 ﾱ 2) minutes. Keep plate covered in the dark and level during incubation.
7. Wash and blot the wells as described in Steps 4 and
5.
8. Pipette 100 µL of TMB Substrate Solution into each well.
9. Incubate in the dark at RT for precisely ten (10) minutes.
10. After ten minutes, add 100 µL of Stop Solution to each well.
11. Determine the absorbance at 450 nm of the contents of each well. Zero the plate reader to air. The absorbance of the final reaction mixture can be measured up to 2 hours after the addition of the Stop Solution. However, good laboratory practice dictates that the measurement be made as soon as possible.

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, although a second order polynomial (quadratic) or other curve fits may also be used.
   3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at IgA 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 pipettings, 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 Dog IgA Calibrator.
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-IgA-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-dog IgA coated wells are stable until the expiration date, and should be stored at 4°C in the sealed foil pouch with desiccant pack.
8. Dog IgA Calibrator: Long Term Storage : Upon receipt, aliquot the calibrator and store them frozen. They will be stable until expiration date.

Haltbarkeit: The expiry date is stated on the label.

Details zu IgA

Target: IgA

Substanzklasse: Antibody