Anti-Phospholipid IgG/IgM ELISA Kit

Catalog No. ABIN996875

Product Details

Target: Anti-Phospholipid IgG/IgM

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0-100 U/mL

Minimum Detection Limit: 0 U/mL

Application: ELISA

Sample Type: Serum

Analytical Method: Quantitative

Sensitivity: 0.5 U/mL

Material not included: 1. Microplate reader capable for endpoint measurements at 450 nm
2 . Multi- Channel Dispenser or repeatable pipet for 100 μL
3. Vortex mixer
4. Pipets for 10 μL, 100 μL and 1000 μL
5. Laboratory timing device
6. Data reduction software

Application Details

Sample Volume: 100 μL

Assay Time: 1 h

Plate: Pre-coated

Protocol: 1. Do not use kit components beyond their expiration dates.
2. Do not interchange kit components from different lots.
3. All materials must be at room temperature (20-28 °C).
4. Have all reagents and samples ready before start of the assay. Once started, the test must be performed without interruption to get the most reliable and consistent results.
5. Perform the assay steps only in the order indicated.
6. Always use fresh sample dilutions.
7. Pipette all regents and samples into the bottom of the wells.
8. To avoid carryover contaminations change the tip between samples and different kit controls.
9. It is important to wash microwells thoroughly and remove the last droplets of wash buffer to achieve best results.
10. All incubation steps must be accurately timed.
11. Control sera or pools should routinely be assayed as unknowns to check performance of the reagents and the assay.
12. Do not re-use microplate wells. For all controls, the respective concentrations are provided on the labels of each vial. Using these concentrations a calibration curve may be calculated to read off the patient results semi-quantitatively.

Reagent Preparation:

1. Distilled or deionized water
   2. Graduated cylinder for 100 and 1000 mL
   3. Plastic container for storage of the wash solution A mixture of highly purified Cardiolipin, Phosphatidyl Serine, Phosphatidyl Inositol, Phosphatidic Acid and human B2-Glycoprotein I is bound to microwells. Antibodies against these antigens, if present in diluted serum or plasma, bind to the respective antigens. Washing of the microwells removes unspecific serum and plasma components. Horseradish peroxidase (HRP) conjugated anti-human IgG or IgM immunologically detectst the bound patient antibodies forming a conjuagate/antibody/antigen complex. Washing of the microwells removes unbound conjuagate. An enzyme substrate in the presence of bound conjugate hydrolyzes to form a blue color. The addition of an acid stops the reaction forming a yellow end-product. The intensity of this yellow color is measured photometrically at 450 nm.

Sample Preparation:

1. Collect whole blood specimens using acceptable medical techniques to avoid hemolysis.
   2. Allow blood to clot and separate the serum by centrifugation.
   3. Test serum should be clear and non-hemolyzed. Contamination by hemolysis or lipemia is best avoided, but does not interfere with this assay.
   4. Specimens may be refrigerated at 2-8 °C for up to five days or stored at -20 °C up to six months.

Assay Procedure:

1. Place the desired number of coated strips into the holder.
   Moderate positive: 40 - 70 SAU PRE-WASH Coated Wells - Repeat washing three times with washing buffer.
   High positive: > 70 SAU Prepare 1:101 dilution of test samples by adding 5 μL of the sample to 500 μL of Sample Diluent. Mix well. Do not dilute 1:101 prediluted Calibrators & Controls. A positive result suggests the possibility of certain autoimmune disease thrombolic disorders. A negative result indicates no beta2 GP1 IgA antibody or levels below the detection limit of the assay. Dispense 100 μL of diluted sera and prediluted calibrators & controls into the appropriate wells. Tap the holder to remove air bubbles from the liquid and mix well. Incubate for 30 minutes at room temperature. Remove liquid from all wells. Repeat washing three times with washing buffer. Dispense 100 μL of enzyme conjugate to each well and incubate for 30 minutes at room temperature. Remove enzyme conjugate from all wells. Repeat washing three times with washing buffer. Dispense 100 μL of TMB Chromogenic Substrate into each well and incubate for 15 minutes at room temperature. Add 100 μL of Stop solution to stop reaction.
   2. Dispense 20 mL of standard, specimens, and controls into appropriate wells.
   3. Dispense 100 mL of zero buffer into each well.
   4. Thoroughly mix for 10 seconds. It is very important to have complete mixing in this setup.
   5. Incubate at room temperature (18-22 °C) for 30 minutes.
   6. Remove the incubation mixture by flicking plate content into a waste container.
   7. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets. a
   9. Dispense 150 mL of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
   10. Incubate at room temperature for 30 minutes.
   11. Remove the incubation mixture by flicking plate contents into a waste container.
   12. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
   14. Dispense 100 mL TMB substrate into each well. Gentle mix for 5 seconds.
   15. Incubate at room temperature for
   2. minutes.
   16. Stop the reaction by adding 100 mL of stop solution to each well.
   17. Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
   18. Read optical density at 450 nm with a microtiter reader within 15 minutes.
   
   Important Note:
   The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

Calculation of Results:

Quality Control This test is only valid if the optical density at 450 nm for Positive Control (1) and Negative Control (2) as well as for the Standards A and F complies with the respective range indicated on the Quality Control Certificate enclosed to each test kit! If any of these criteria is not fulfilled, the results are invalid and the test should be repeated. Calculation of results For the Anti-Phospholipid Screen test a 4-Parameter-Fit with lin-log coordinates for optical density and concentration is the data reduction method of choice. Recommended Lin-Log Plot First calculate the averaged optical densities for each calibrator well. Use lin-log graph paper and plot the averaged optical density of each calibrator versus the concentration. Draw the best fitting curve approximating the path of all calibrator points. The calibrator points may also be connected with straight line segments. The concentration of unknown may then be estimated from the calibrator curve by interpolation. Calculation Example The figures below show typical results for Anti-Phospholipid Screen. These data are intended for illustration only and should not be used to calculate results from another run.
In a normal range study with serum samples from healthy blood donors the following ranges have been established with the anti-Phospholipid Screen test: Anti-Phospholipid-Ab IgG [GPL U/mL] IgM [MPL U/mL] normal: < 10 < 10 elevated: > 10 > 10 Positive results should be verified concerning the entire clinical status of the patient. Also every decision for therapy should be taken individually. It is recommended that each laboratory establishes its own normal and pathological ranges of antiPhospholipid antibodies. This kit is intended for Reserach Use only. Not for use in diagnostic procedures.
Calculate the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Constructed a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ng/mL on graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis. Use the mean absorbance values for each specimen to determine the corresponding concentration of AFP in ng/mL from the standard curve. Example of standard curve Results of typical standard run with optical density reading at 450 nm shown in the Y-axis against AFP concentrations shown in the X-axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve. Expected values and sensitivity In high-risk patients, AFP values between 100 and 350 ng/mL suggest a diagnosis of hepatocellular carcinoma, and levels over 350 ng/mL usually indicate the disease. Approximately 97 % of the healthy subjects have AFP levels less than 8.5 ng/mL. It is recommended that each laboratory establish its own normal range. The minimum detectable concentration of AFP by this assay is estimated to be 2.0 ng/mL.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Expiry Date: 12 months

Target Details

Target: Anti-Phospholipid IgG/IgM

Target Type: Antibody