Indirect Enzyme
Immunoassay for the detection of human antibodies against infectious
agents
In vitro use
Dr. Julio Moran, Laboratorien, Lohwisstrasse 32, CH-8123
Ebmatingen, Schweiz www.jmlab.ch
Indirect enzyme immunoassay
for the detection of antibodies to infectious agents in human serum and plasma.
The enzyme immunoassay is intended for testing individual specimens, not pooled
specimens. In vitro diagnosticum, only to be used for in vitro diagnostic
purposes by correspondingly educated laboratory personnel. The test can be
processed manually or automatically.
The barcode identification of each single reagent ensures their
correct identification and an accurate
automatic processing of the test.
Instructions for use 1
Adhesive foils 2
Bag with additional bar coded labels (1 each), to
identify additional vials for
the preparation of ready to use reagents (Nr.5/6, 7, 8/9, 11) 1
1 Microplate strips , antigen coated (12 x 8 break apart
wells) 2
bags
2 Negative Control, human, ready to use (light green) 1
1.5 ml
3 Differential Control, human, ready to use (orange) 1 3 ml
4 Positive Control, human, ready to use (red) 1
1.5 ml
5 Diluent buffer (blue) 2
20 ml
6 Additive for diluent buffer 2 2 ml
7 Anti-human Ig- G, M, A, MA Conjugate, ready to use
(blue, yellow,green,red) 1
12 ml
8 Substrate buffer with Substrate 1
15 ml
9 Chromogen-concentrate
(21x conc TMB in DMSO, corrosive) 1 0.75 ml
10 Washing solution concentrate (25x conc) 2
20 ml
11 Stopping solution (sulfuric acid, max. 0.2 M, corrosive) 1
15 ml
Deionized
water, graduated cylinders 1000 ml, 500 ml, 250 ml, 100 ml. Pipettes with a
fixed or variable volume of 10, 100, 200 and 1000 microliters. 8-channel
pipettes with variable volume of 100 or 200 microliters.
Additional
vials (10 ml, 20 ml) for making the ready to use solutions.
Tubes
with low protein absorption (e.g. polypropylene, polyethylene or glass) for
making sample predilutions if necessary.
Incubator 37 °C +/- 1 °C (dry incubator, make sure to
correctly seal the wells with the adhesive foil to prevent evaporation which
may lead to erroneous results). Timer.
Washing
device: using manual or automatic washing devices optimise settings according
to the manufacturer’s instructions for use, so that the validation criteria of
the test are fulfilled.
Mcirotiterplate
reader having a 450 nm filter, reading at the measuring wavelength (450 nm) and
using a reference wavelength a filter
for 620 to 650 nm is recommended.
Fully
automated processing of the test with
processing systems utilizing barcode identification is possible.
DESCRIPTION STORAGE STABILITY COMMENTS
Closed components of the test kit
2...8 °C until expiry date
Opened Microplate strips 2...8
°C 6 weeks keep storage bag tightly closed
avoiding high humidity
Opened components No. 2, 3, 4, 5, 6, 7 2...8
°C 12 weeks avoid Temperature stress and
contamination
Opened substrate buffer (No.8) 2...8 °C 12 weeks avoid direct exposure to light
Opened TMB/Chromogen solution 21x conc (No.9) 2…8 °C 12
weeks avoid direct exposure
to light
Specimen diluent (No.5 + No.6), ready to use 2…8 °C 12
weeks prepare only the
necessary volume
and
avoid contamination
TMB-Chromogen/Substrate solution (No.8 + No.9), 2…8 °C max. 24
h prepare only the necessary
volume
ready to use and
avoid direct exposure to light
Washing solution, ready to use 2…8 °C 12 weeks use only a clear solution
20…25 °C max. 2 weeks use only a clear solution
Stopping solution 2…8 °C until expiry date
The test should only be performed by properly
trained professional laboratory staff.
All reagents must have reached room temperature
(22...25 °C) prior to start performing
the test run.
Incubation periods: Specimens and controls 30
min. at 37 °C, Conjugate 30 min. at 37°C and TMB-Chromogen / Substrate solution
10 to 20 min. at room temperature (22…25 °C). Adaptation of incubation periods
to specific internal laboratory needs is possible, according to GLP they should
be validated.
PREPARATION
OF REAGENTS / PREPARATION OF
TESTPROTOCOL
SPECIMEN
DILUTION: 1/21 dilution
Performing multiple testing of specimens a corresponding volume is
prepared in a separate tube or plate:
0.100 ml OF THE SPECIMEN DILUTION ARE
PIPETTED
Pipetting specimens directly in the plate dispense first 0.200 ml of
specimen diluent and then add 0.010 ml
specimen
0.200 ml OF SPECIMEN DILUENT + 0.010
ml SPECIMEN
0.100 ml OF THE CONTROLS ARE PIPETTED
INCUBATION
OF SPECIMENDILUTIONS AND CONTROLS 30 min. at 37 °C
WASH 4x
PIPETTING OF THE CONJUGATE (0.100 ml)
INCUBATION WITH CONJUGATE 30 min. at 37°C
WASH 4x
PIPETTING OF THE TMB-CHROMOGEN-SUBSTRATE SOLUTION (0.100 ml)
INCUBATION
WITH TMB-CHROMOGEN-SUBSTRATE SOLUTION
(10 …20 min at room temperature,
ca. 20…25 °C)
PIPETTING OF THE STOPPING SOLUTION (0.100 ml)
PHOTOMETRIC MEASUREMENT at 450nm (Ref:630 nm)
TESTVALIDATION
/ EVALUATION
Validation:
Results obtained in absorbance units
(extinction units, O.D. units) for the
controls are used if the values of the differential control are higher than
0.080 and lower than 1.000 (optimally between 0.200 and 0.600) and the
deviation of the values obtained for the differential control falls within +-
20 % of the mean value. Additionally the corresponding index value of the
negative control must be < 0.6 and the corresponding index value of the
positive control must be > 1.4.
These criteria apply to all our systems.
absorption
at 450nm of the corresponding control
Index
value of the controls =
--------------------------------------------------------------------------------
Mean
absorption at 450nm of the differential control
Example of a validation: mean value of the absorption f the differential
control 1. value: 0.280, 2. value:
0.320 Mean value : 0.300
controls O.D.-
value 450 nm Index
Absorption (O.D. value) of the negative control
(Nr. 2)......0.100 0.100 / 0.300 = 0.333
Absorption (O.D. value) of the differential control
(Nr. 3) .....0.280 0.280 / 0.300 = 0.933
Absorption (O.D. value) of the differential
control (Nr. 3) .....0.320 0.320 / 0.300 = 1.067
Absorption (O.D. value) of the positive control (Nr. 4)......0.600 0.600 / 0.300 = 2.000
Are the values obtained within the range of the
validation criteria, then the test run is valid, and evaluation can be
performed.
If the validation criteria are not met, then
corrective measures may be applied to the obtained vales before repeating the
test run.
Corrective
Measures:
Are the OD-values obtained too high, then a correction factor or a
general dilution step followed by a volume reduction of the colour solution in
each well (samples and controls) may be applied, in this way the colour
intensity may be reduced to fit the validation criteria (e.g.: factor 0.5, will
reduce all OD-values by a half, a dilution step 1in 2 will also reduce the
OD-values by half) . Alternatively
performing the next test run the reaction time with the chromogen/substrate
solution can be shortened from e.g. 10 to 20 min to 5 to 10 min. to remain within the validation range. Are
the obtained O.D.-values still too high, then the chromogen concentration in
the chromogen/substrate solution must be reduced: dilute the concentrated
chromogen solution 1 in 41 (0.025 ml reagent No. 9 in 1 ml reagent No. 8) in
stead of 1 in 21 and incubate between 5 to 15 min.
Are the obtained results too low then it is
possible to introduce a simple multiplication of the obtained results by a
factor (2 to 3) to reach the range of the validation criteria. Alternatively
performing the next test run the reaction time with the chromogen/substrate
solution can be extended from e.g. 10 min to 20 min or to 30 min. to reach the
validation range (see corrective measures in the detailed test procedure).
Should these possible corrective measures still
not lead to acceptable results, then the test run has to be repeated.
Evaluation of test results can be performed if
the validation criteria apply. Evaluation of
the results for each specimen is done after calculating the Index value
for each single specimen. Calculation of the index value corresponds to a
normalization of the results against the value obtained for the differential
control in each single test run and may be assigned as a ‘test reference
value’. The Index value is obtained by dividing the absorption value (extinction,
O.D. value) of each single specimen by the mean value of the differential
control.
Absorption
at 450 nm of a specimen
Index = -------------------------------------------------------------------------------- (Index of a specimen)
Mean Absorption at 450 nm of the differential
control
Index values (Test reference values) higher
than 1.00 are scored reactive and indicate a presence of IgG antibodies, Index values lower than 0.90 are scored non
reactive and indicate an absence of IgG antibodies. Index values between 0.90 and 1.00 are scored questionable. For
weakly reactive results it is recommended to consider a confirmatory test or to
request a second specimen 10 to 14 days later to be tested in the same test run
with the first specimen.
Example of a qualitative evaluation
Qualitative evaluation is done according to the
reactivity of the differential control. All specimens giving Index values
higher than that of the differential control are considered as reactive and all
giving lower Index values are considered as non reactive. The entire Index
range may be divided in ranges with increasing reactivity and to these ranges a
diagnostic meaning may be assigned. The higher the reactivity the higher the
diagnostic meaning.
Mean value of the differential control : 1.
value : O.D. 0.280, 2. value : O.D. 0.320, Mean value:
O.D. 0.300
Specimen O.D. 450 nm Index/ Test reference value
Index Evaluation
Ranges
range
Spec. No. 1........0.080 0.080 / 0.300 = 0.266 < 0.900 non
reactive
Spec. No. 2........0.280 0.280 / 0.300 = 0.933 0.900-1.000 border line
Spec. No. 3........0.350 0.350 / 0.300 = 1.167 1.000-1.500 weakly reactive
Spec. No. 4........0.500 0.500 / 0.300 = 1.667 1.500-2.000
reactive
Spec. No. 5........0.700 0.700 / 0.300 = 2.333 2.000-3.000
highly reactive
Spec. No. 6........1.000 1.000 / 0.300 = 3.333 3.000-5.000 very
highly reactive
Example of a quantitative evaluation after
introduction of relative units
For clinical reports quantitative results in
relative units are usually requested to better assess and assign the results
obtained. For this purpose the simplest way is to multiply the Index value with
a simple factor and assign the new range of values a new range of units. It is
to be considered that these relative units are also based on a logarithmic
scale.
Example: multiplying the Index values of the
specimens in above table by 10 gives the new unit values (logarithmic scale):
Relatonship between O.D.values, Index values
and unit values for the above mentioned results
Spec.
No.1 Spec. No.2 Spec. No.3 Spec. No.4 Spec.
No.5 Spec. No.6
O.D.values: 0.080 0.280 0.350 0.500 0.700 1.000
Index values: 0.266 0.933 1.167 1.667 2.333 3.333
Units values: 2.66 9.33 11.67 16.67 23.33 33.33
Further mathematical evaluation methods of the
results, like using a standard curve (with serum dilutions) as a
reference, or with the help of the
<one point quantification> are also possible. However it has to be kept
in mind that all these additional evaluation methods use one common basic
operation: calculating a reference value of the basic reactivity with at least
one standard before further mathematical transformation (logarithmic,
exponential, polynomial, 4 PL Model,
etc.) is done to obtain the corresponding relative units.
The scales of the relative units found are also
divided in reactivity ranges with increasing reactivity, that can be related to
an increasing probability of a diagnostic indication
In principle however all these evaluation methods
operate with the same originally measured values (absorption, extinction, O.D.
value) and corresponding differentiating reactivity ranges.
The probability to assign a diagnostic significance
to a given reactivity increases with increasing absorption value, or increasing
Index value or increasing value of relative units.
EXAMPLE:
Specimen O.D. 450 nm Index Index Range Relative Units Relative Evaluation
Diagnostic
(e.g.:
Index x 10) Units Range Ranges Significance
Spec. No.
1........0.080 0.266 < 0.900 2.66 < 9 non
reactive - - -
Spec. No. 2........0.280 0.933
0.900-1.000 9.33 9 –10 border
line - - / +
Spec. No. 3........0.350
1.167 1.000-1.500 11.67
10 –15 weakly reactive
- / + +
Spec.
No. 4........0.500
1.667 1.500-2.000 16.67
15 –20 reactive + + +
Spec.
No. 5........0.700
2.333 2.000-3.000 23.33
20 –30 highly reactive
+ + + +
Spec.
No. 6........1.000
3.333 >3.000 33.33 >30 very
highly reactive + + + + +
In general the presence of IgG antibodies
indicates a past infection or vaccination. The detection of IgG antibodies during
the course of an infection may indicate a current infection, if the results of
a parallel determination of two
specimens from the same patient, taken 10 to 14 days apart, indicate a
seroconversion (conversion from negative to positive).
It is to be considered that in the early stage
of a seroconversion the results obtained may still fall under the values of the
differential control.
Borderline and weakly reactive results should
be retested together with an additional sample drawn 10 to 14 days apart. If no
differences in reactivity are detected no evidence for a current infection may
be assigned, if clear increments in reactivity are detected, support for a
current infection may be indicated.
Very high IgG reactivities may indicate the
peak of the acute phase of a current infection.
The simultaneous detection of IgM- and
IgA-antibodies during a seroconversion very strongly support a current infection.
Interpretation of serological results should
always only be done together with clinical data.
The presence of interfering factors such as
Rheumatoid Factor (RF) may lead to false positive results for IgM and also
IgA.. Testing for the presence rheumatoid factor and a preabsorption with
RF-Absorbent before performing the test run is recommended.
(RF-Absorbent can also be additionally supplied
by us and ordered under REF RFA-192 (10 ml, for 100 Tests) ).
Literature
recommended for further reading:
American
Public Health Association Bookstore
Clinical Microbiology Procedures Handbook
Communicable Disease Control and Laboratory
Procedures