INTENDED USE

INTRODUCTION
Anti-thrombin III is a small glycoprotein produced by the liver.  It is a major inhibitor of the coagulation cascade and inhibits virtually all of the coaquiation enzymes, including thrombin.  Deficiency of Anti-thrombin III, either genetic or acquired can lead to thromboembolic disease.
PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1.  In this assay the AT-III present in the sample reacts with the anti-AT-III antibody, which has been adsorbed to the surface of polystyrene microtiter wells.  After the removal of unbound sample proteins by washing, anti-AT-III antibody conjugated with horseradish peroxidase (HRP) is added.  This HRP-conjugated antibody forms a complex with the previously bound AT-III.  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 AT-III in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of AT-III in the test sample.  The quantity of AT-III in the test sample can be interpolated from the calibration curve constructed from the calibrators and corrected for sample dilution. Figure 1.
Unbound Anti-AT-III-HRP Removed TMB Substrate Added Determine Bound Enzyme Activity
COMPONENTS
Diluent Concentrate 1. 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 soulution. 3.   Enzyme-Antibody Conjugate Concentrate One vial containing 150 μL of a 100X concentrated affinity-purified anti-rat AT-III 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-rat AT-III. 7.  Rat AT-III Calibrator One vial containing a lyophilized Rat AT-III Calibrator. 8.   Positive Control One vial containing 50 μL of serum with 0.1% sodium azide.  See the Control Certificate for the concentration.
MATERIALS REQUIRED BUT NOT PROVIDED
. 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 . .
PRECAUTIONS
1. 2i 3. 4. 5. 6. 7. Read the instructions carefully before beginning the assay. This kit is for research use only. Great care has been taken to ensure the quality and reliability of this product.  However, it is possible that in certain cases, unusual results may be obtained due to high levels of interfering factors. Preservatives ➤ Positive Control contains 0.1% sodium azide. No additives or preservatives are necessary to maintain the integrity of the specimen.  Avoid azide contamination. Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction. Other precautions: ▷ Do not interchange kit components from different lots. ▷ Do not use kit components beyond the expiration date. ▷ Protect reagents from direct sunlight. ▷ Do not pipette by mouth. ▷  Do not eat, drink, smoke or apply cosmetics where reagents are used. ▷ Avoid all contact with the reagents by using gloves. ▷ Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water after contact.
REAGENT PREPARATION
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. Rat AT-III Calibrator
Add 1.0 mL of distilled or de-ionized water to the Iyophilized Rat AT-III Calibrator and mix gently until dissolved.  The calibrator is now at a concentration of 1.2 μg/mL (the reconstituted calibrator should be aliquoted and frozen if future use is intended). Rat AT-III Calibrators need to be prepared immediately prior to use (see chart 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.
STORAGE AND STABILITY
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 Rat AT-III 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-AT-III-HRP conjugate should be stored at 4°C and diluted immediately prior to use.  The working conjugate solution is stable for up to eight hours at 4°C. 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-rat AT-III coated wells are stable until the expiration date, and should be stored at 4°C in the sealed foil pouch with desiccant pack. 8. Rat AT-III Calibrator The lyophilized Rat AT-III 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 eight hours at 4°C. 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.
INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expected values.
SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture and the serum separated from the cells, after clot formation, by centrifugation. For plasma samples, blood should be collected into a container with an anticoagulant and then centrifuged. Care should be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay immediately or aliquot and store samples at -20°C.  Avoid repeated freezing/thawing. For any sample that might contain pathogens, care must be taken to prevent contact with open wounds.  No additives or preservatives are necessary to maintain the integrity of the specimen.  Avoid azide contamination.
ASSAY PROTOCOL
Dilution of Serum or Plasma Samples
Due to the high-sensitive nature of the assay each serum or plasma sample should be diluted before use for a normal assay.  A 1:10,000 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 1:10,000 dilution of sample, transfer 5 μL of sample to 495 μL of 1X Diluent.  This gives you a 1:100 dilution. Next, dilute the 1:100 samples by transferring 5 µ L to 495 µ L of 1X Diluent.  You now have a 1:10.000 dilution of your sample.  Mix thoroughly at each stage.
Procedure
Bring all reagents to RT before use. 1. Bring all reagents to RT before use. 2. Pipette 100 μL of Calibrator 0 (0.0 ng/mL) in duplicate Calibrator 1 (15.6 ng/mL) in duplicate Calibrator 2 (31.25 ng/mL) in duplicate Calibrator 3 (62.5 ng/mL) in duplicate Calibrator 4 (125 ng/mL) in duplicate Calibrator 5 (250 ng/mL) in duplicate Calibrator 6 (500 ng/mL) in duplicate Calibrator 7 (1,000 ng/mL) in duplicate 3. Pipette 100 μL of diluted Positive Control (in duplicate) into pre designated wells. 4. Pipette 100 μL of diluted sample (in duplicate) into pre designated wells. 5. Incubate the Microtiter Plate at 22°C (RT) for sixty (60 ± 2) minutes.  Keep plate covered and level during incubation. 6. Following incubation, aspirate the contents of the wells. 7. 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. 8.   Pipette 100 μL of appropriately diluted Enzyme-Antibody Conjugate to each well.  Incubate at 22°C (RT) for thirty (30 ± 2) minutes. Keep plate covered in the dark and level during incubation. 9. Wash and blot the wells as described in Steps 6 and 7. 10. Pipette 100 μL of TMB Substrate Solution into each well. 11. Incubate in the dark at RT for precisely ten (10) minutes. 12. After ten minutes, add 100 μL of Stop Solution to each well. 13. 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.
RESULTS
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 AT-III concentration in original sample.
PERFORMANCE CHARACTERISTICS
In accord with good laboratory practice, the assays for specific AT-III require meticulous quality control.  Each laboratory should use routine quality control procedures to establish inter- and intra-assay precision and performance characteristics.
LIMITATION OF THE PROCEDURE
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.
FOR RESEARCH USE ONLY
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