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.
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
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
PIPETTING OF THE CONJUGATE (0.100 ml)
INCUBATION WITH CONJUGATE 30 min. at 37°C
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
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.
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
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.
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: