NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE

Wednesday, May 05, 2004

Description
Neopterin [D-erythro-neopterin] belongs to the group of pteridines.
Neopterin as well as other pteridines are derived in vivo from guanosinetriphophate
(GTP). The enzyme GTP-cyclohydrolase-I catalyses this
reaction in monocytes and macrophages (fig.1). Neopterin is exclusively
excreted by activated macrophages (fig.2), and can be determined in serum,
plasma and other body fluids by immunoassay.
The principal of the ELItest Neopterin test procedure is that of an Enzyme
immunoassay (EIA) based on the competition of unlabelled neopterin of the
standards, controls samples, patients samples and enzyme conjugated
(Neopterin/alkaline phosphatase conjugate) for the binding sites of the
anti-neopterin-antibodies (polyclonal, sheep). An antigen-antibody
complex is formed and bound to the solid phase (coated well). The
following intensive washing ensures the complete removal of all unbound
components. The addition of 4-nitrophenyl-phosphate substrate solution
starts the enzyme reaction, where the alkaline phosphatase being part of
the neopterin/enzyme conjugate catalyses the separation of the phosphate
of the 4-nitrophenyl-phosphate, thus setting free the yellow 4-
nitrophenol. Pipetting of sodium hydroxide stops the enzymatic reaction.
The intensity of the color (measured in optical densities or OD) depends
on the amount of enzyme bound in the wells at a constant reaction time and
consequently is inversely proportional to the neopterin concentration in
the patient sample. Thus high neopterin values correspond to low amounts
of optical density. The optical density is measured by a microtiter plate
reader with a wavelength of 405 nm. For calculation of results a standard
curve is created (optical Density versus concentrations of neopterin
standards), allowing the determination of the neopterin concentrations
within the patient samples.The following clinical indications for determination of neopterin can be
emphasized:
@ Monitoring of HIV infected individuals
@ Surrogate marker for the effects of antiviral therapy in HIV
infected Individuals.Screening of donated blood and bone marrow for avoidance of HIV
infection.
@ Testing of infants born to HIV+ mothers.
@ Follow up of transplant recipients for rejection.

Procedure
ASSAY PROCEDURE:
5.1 General Remarks:
5.1.1 Before performing the assay, samples and assay kit should be
brought to room temperature.
5.1.2 All Standards should be run with each series of unknown
samples.
5.1.3 Each Standard and sample should be assayed in duplicate each
time the test is performed.
5.1.4 Standards should be subject to the same manipulations and
incubation times as the samples being tested.
5.2 Step by step ELItest Neopterin procedure:
Do not interrupt working procedure. Keep the order and the
incubation times constant for sample. The wells on the plate should
always be filled in the same order.
5.2.1 Pipette 50 l standards (increasing concentrations), 50 l
control and 50 l serum samples into the uncoated microtiter
plate.
5.2.2 Pipette 150 l of the enzyme conjugate into the uncoated
microtiter plate containing standards, controls and samples.
It is recommended to ensure a good mixture after pipetting by
intensive, brief shaking of the uncoated microtiter plate.
5.2.3 Transfer an aliquot of 150 l of mixture 5.2.2 into the
neopterin-antibodies coated microtiter plate. It is
recommended to use a multi-channel pipette.
5.2.4 Incubate the microtiter plate for 2 hours at room temperature
in the dark (using black cover sheet).
5.2.5 Afterwards aspirate or decant the liquid completely from the
wells, Wash wells with 350 l washing buffer. Aspirate or
decant the wells once again completely and repeat this step
three more times.
5.2.6 After the last wash remove any remaining droplets by tapping
the plate on blotting paper observing carefully that no
residual droplets remain in the wells.
5.2.7 Using multi-channel pipette, pipette 100l of the substrate
(4-nitrophenyl-phosphate) into all wells.
5.2.8 Incubate the microtiter plate once again for 30 10 minutes
at room temperature. This reaction also can be performed
under normal daylight.
5.2.9 Using multi-channel pipette, pipette 100 l of stop solution
(2N sodium hydroxide) into all wells to stop the enzyme
reaction. Pipetting of stop solution has be done in the same
order as for the substrate.
5.2.10 Measurement of the optical density (OD) is carried out
in a photometer at wavelength of 405 nm.
6. CALCULATION:
6.1 Calculate the average absorbance values (A ) for each set of 405
duplicate Standards, samples and controls.
6.2 If individual absorbance values differ by more than 15% from the
corresponding mean value, the result is considered suspect and the
sample should be re-assayed.
6.3 The mean absorbance value of the controls should be less than 10%.
6.4 Construct a standard curve by plotting the mean absorbance for each
Standard on the vertical (Y-axis, logarithmic) axis versus the
corresponding Neopterin concentration on the horizontal (X-axis,
linear) axis, thus creating a standard curve by means of 6 points
obtained.
6.5 Using the mean absorbance value for each sample, determine the
corresponding concentration of neopterin from the standard curve.
Find the absorbance value on the Y-axis and extend a horizontal line
to the curve. At the point of intersection, extend a vertical line
to the X-axis and read the neopterin concentration for the unknown
sample.
6.6 If the samples have been diluted, the concentration determined from
the standard-curve must be multiplied by the dilution factor.
6.7 The sigmoid function available in AssayZap software on Apple
computer or Windows version 2.5 by Biosoft INC Tel:(314) 524-8029)
is suitable for computer assisted analysis of Elitest neopterin.
7. QUALITY CONTROL:
7.1 In accord with modern management practice, quality control and
assurance must be regarded as a collaborative exercise with active
and committed participation of all personnel concerned, from the
most neophyte to the most qualified. Low or zero rejection rates
require corrective actions to be taken before actual problems arise,
which is impossible without the full participation of those most
closely involved. (Precision of pipettes, diluters, readout devices
and good operator technique.)
7.2 The samples used for internal or external quality control purposes
must resemble the patient samples. For this purpose CIRID at UCLA
recommends that aliquots from two large number of aliquots of inhouse
reference samples should be prepared. One set of reference
samples should be normal or slight abnormal and another set should
be distinctly abnormal. The number of aliquots in these separate
quality control pools must be available in sufficient quantity and
sufficient stability for extensive serial laboratory testing.
7.3 One aliquot from each of the two In house reference should be used
as an internal laboratory control in each run. Reference samples
should be run in triplicates.
7.4 Kit (range from 5 to 60 nmol/l) and In house controls must be
routinely assayed as unknown samples to measure assay variability.
The results of all tests should be serially plotted to monitor the
performance of the kits and laboratory staff.
7.5 Repeat testing on subsequent assay day of 15-20% patient samples is
also recommended. It used to measure inter-assay precision.
7.6 Proficiency testing:
7.6.1 Participate in a national inter-laboratory proficiency program
as available.
8. EXPECTED VALUES:
Available data indicates that 97% of the values obtained from blood donors
are less than 10 nmol/l. Thus a preliminary cut-off of 10 nmol/l can be
recommended. This corresponds well to the investigation of Honlinger et
al 1989 using RIA for determination of neopterin. It is recommended that
each laboratory establish its own expected range. Serum and plasma
samples from apparently healthy individuals were evaluated for Nepoterin
with IMMUtest Neopterin RIA kit and ELItest Neopterin EIA kit Henning
Berlin GMBH in our laboratory (CIRID at UCLA).
CIRID at UCLA Laboratory Reference for Normal value using IMMUtest
Neopterin RIA test kit.
Serum or Plasma No. Mean * SD Median * 10th % 90th %
Adult 65 5.97 2.93 5.53 3.60 7.77
Male/Female
CIRID at UCLA Laboratory Reference for Normal Value using ELItest Neopterin EIA
test kit.
Serum or Plasma No. Mean * SD Median * 10th % 90th %
Adult Male/ 30 5.41 2.11 5.25 3.76 7.34
Female
* nmol/l
9. PROCEDURE NOTE:
9.1 The kit should not be used beyond expiration date.
9.2 Since assay condition may vary from assay to assay, a standard curve
must be established for every assay run.
9.3 Completely aspirate well contents before dispensing fresh wash
solution.
9.3.1 Fill with wash buffer to the top of the well for each wash
cycle (approx. 250 L).
9.3.2 Do not allow wells to sit uncovered or dry for extended
periods between incubation steps.
9.4 Check the data for:
9.4.1 The Coefficient of Variation (%) of Standards must be less
than 10%.
9.4.2 The Coefficient of Variation (%) of Controls (triplicate) must
be less than 15%.
9.4.3 Random retest of 15-20% of the samples must vary by less than
15% of original value.
9.5 The results are checked and verified by the section supervisor and
reported to the data manager for data entry. Final data print-outs
are checked by the Associate Director or Director of CIRID before
results are sent out.
10. LIMITATION OF THE METHOD:
10.1 This kit is for Research use only. It is not for Diagnosis of
diseases.
10.2 For values higher than the highest standard should be diluted with
Physiological sodium chloride solution, and repeat the assay.
11. METHOD VALIDATION:
11.1 Minimum Detectable Concentration (MDC): The MDC of ELItest
Neopterin EIA test kit is 2.0 nmol/l.
11.2 Precision: (CIRID at UCLA Data)
11.2.1 Intra-assay precision was determined from mean of 8
replicates of each sample.
Sample Mean (nmol/l) SD Coeff. of
Variation(%)
Serum-CIRID-1 5.34 0.19 3.6
Serum-CIRID-2 17.82 0.54 3.0
11.2.2 Inter-assay precision was determined from the mean of
triplicates of each sample for six different assays.
Sample Mean SD Coeff. of
(nmol/l) Variation(%)
Serum-CIRID-1 4.56 0.60 13.0
Serum-CIRID-2 17.63 0.63 3.61
Kit-control#1 7.19 0.69 9.60
Kit-control#2 65.42 3.36 5.14
11.3 Specificity:
The test kit is specific for Neopterin.
12. GENERAL INFORMATION ABOUT THE PRODUCT:
Name: Elitest Neopterin ELISA Test Kit
Cat #: H-650
Manufactured by: B.R.A.H.M.S. Diagnostica GMBH (Berlin Germany)
Distributor by: Polymedco Inc. (责任编辑：泉水)

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NEOPTERIN ENZYME IMMUNOASSAY (EIA) TEST PROCEDURE