NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE(2)

Recipes
2. SPECIMENS:
2.1 Patient Preparation:
There is no specific instruction for the patient, but it is better
to ask the patient to avoid fatty food for at least two hours before
blood draw.
2.2 Type of Specimens:
Specimens should not be exposed to direct sunlight.
2.2.1 Blood Collection:
2.2.1.1 Serum:
-Blood collected by venipuncture into a serum
collecting tube in accordance with approved
standard.
Allow specimen to clot (0.5-1 hour) at room
temperature.
- Separate the serum by centrifugation at
2000 RPM for ten minutes.
- Aliquot the serum into 1.8 ml labeled
cryovial. Store at -70EC.
- Minimum amount (120 l) and maximum amount
(500 l) of serum required for the
neopterin assay.
- If more than 2 hours elapses before
separation, place the tube in the 4EC
refrigerator.
2.2.1.2 Plasma:
- Blood collected by venipuncture with
approved standard using an appropriate
anticoagulant EDTA, Heparin or Citrate.
- Mix the specimen gently and allow the tube
to stand for 1/2 hours.
- Separate the plasma from the other
components of the blood by centrifugation
(1500 rpm for 15 minutes).
- Aliquot 1.8 of plasma into 1.8 ml of
labeled cryovial. Store at -70EC.
- Minimum amount (120 l) and the maximum
amount (500l) of plasma required for the
neopterin assay.
2.2.2 CSF Collection:
- C.S.F. should be collected into a sterile, screw capped
tube - Centrifuge the
tube at 3000 RPM
for ten minutes
- Pipette CSF out of the tube without disturbing the
pellet, and leave at least 0.5 ml of CSF in the tube.
- Aliquot the CSF into 1.8 ml labeled cryovials and store
at -70EC
- Minimum amount of 200 l and maximum amount of 500 l
CSF needed for neopterin assay
2.3 Specimens Labeling:
- Clearly label the following information on the cryovials
- PID
- Date of draw
- Type of specimen
2.4. Transport and Storage:
2.4.1 On site samples processing and storage:
- Follow the procedure for sample processing
- Store the samples at -70EC until assay on site or ship
to central assay Lab
2.4.2 Ship the frozen samples overnight according to the shipment
rules and regulations for biohazard specimen.
3. MATERIALS USED IN THE ASSAY:
3.1 Materials provided:
3.1.1 ELItest Neopterin test kit provides a plate sufficient for 96
determinations.
3.2 Materials required but not provided:
3.2.1 Micropipettes disposable tips (50 l, 100 l, 200l, and 300
l ).
3.2.2 Volumetric pipettes. For various steps, use of multichannel
pipette (8 channel microwell pipette) is advised.
3.2.3 Distilled or deionized water.
3.2.4 Polypropylene tubes.
3.2.5 Linear graph paper.
3.2.6 Disposable reagent troughs for multi-channel pipette.
3.3 Instrumentation:
3.3.1 Automatic or semi-automatic rinsing/aspiration equipment can
be used to perform the washing steps in the assay procedure.
3.3.2 Titertek Multiskan MCC Microtiter Plate Reader or any brand
with wavelength set at 405 nm. Clean and Calibrate annually.
3.3.3 Rotating platform 700 100 RPM.
3.3.4 Biosaftey Hood:
Technical Safety Services Inc. (TSS) 1-800-877-7742 Test and
certify the biosafety hood annually.
3.3.5 Pipette: Precision pipette of volumes of: 50 l, 100 l, 200
l, 1 ml and 10 ml and Multi-channel pipettes (50-300 l).
Clean and calibrate the pipettes semi-annually. Calibrate
Inc. The Pipette people (1-800-253-7064).
3.4 Reagents:
All reagents in this kit are for in vitro diagnostic use only.
3.4.1 Microtiter plate
1 Microtiter plate for 96 determination with 12 strips each
with 8 wells, coated with anti-neopterin-antibodies
(polyclonal, sheep). Ready for use.
Storage: Refrigerate at 2 to 8EC
Stability: Refer to expiration date on the kit
3.4.2 1 Microtiter plate for premixture of enzyme conjugate and
standards, controls and samples.
3.4.3 Washing solution
1 bottle containing 10.5 ml of concentrate buffer.
Preparation: To be diluted up to 500 ml with distilled
water before use.
Storage: Refrigerate at 2 to 8EC
Stability: Refer to expiration of the kit
3.4.4 Neopterin standards
Neopterin in neopterin-free human serum plus preservative at
the following concentration:
def.; 2; 6.4; 16; 40; 100; 250 nmol neopterin/liter
Storage: Refrigerate at 2-8EC. For prolonged storage
freeze at -20EC.
Stability: Refer to expiration date on kit
3.4.5 Enzyme conjugate 1 vial, Lyophilized neopterin/alkaline
phosphatase conjugate
Preparation: To be diluted with 16 ml Enzyme conjugate
buffer before use.
Storage: Refrigerate at 2-8EC
Stability: Refer to expiration of the kit
3.4.6 Enzyme conjugate buffer
1 vial containing 16 ml of buffer.
Preparation: Ready to use
Storage: Refrigerate at 2-8EC
Stability: Refer to expiration date on the kit
3.4.7 Substrate (4-nitrophenylphosphate)
2 tablets substrate.
Preparation: to be dissolved in 15 ml of substrate
buffer before use
Storage: Refrigerate at 2-8EC
Stability: Refer to expiration date on the kit
3.4.8 Substrate buffer
1 vial containing 15 ml, for dissolving tablets (5.7)
Preparation: Ready to use.
Storage: Refrigerate at 2-8EC
Stability: Refer to expiration date on the kit
3.4.9 Stop solution
1 vial containing 15 ml of 2N sodium hydroxide
Preparation: Ready to use
Storage: Refrigerate at 2-8EC
Stability: Refer to expiration date on the kit
3.4.10 Neopterin control
Neopterin in human serum, plus preservative in two different
concentrations.
Preparation: Ready to use
Storage: Refrigerate at 2-8EC and for prolonged storage
freeze at -20EC.
Stability: Refer to expiration date on the kit
3.4.11 ELItest Neopterin EIA kit instruction Manual.

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NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE(2)