GENERAL TESTPROCEDURE (short version)

DIACHECK Anti IgG, -M, -A, -M/A

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

 

 

A. INTENDED USE

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.

 

 

B. PACKAGE CONTENT (for totally 96 Tests)

 

Nr. DESCRIPTION AMOUNT VOLUME

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

Storage bag for strips 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

 

C. MATERIAL REQUIRED BUT NOT SUPPLIED

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 manufacturers 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.

 

D. STORAGE AND STABILITY

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) 28 C 12 weeks avoid direct exposure to light

Specimen diluent (No.5 + No.6), ready to use 28 C 12 weeks prepare only the necessary volume

and avoid contamination

TMB-Chromogen/Substrate solution (No.8 + No.9), 28 C max. 24 h prepare only the necessary volume

ready to use and avoid direct exposure to light

Washing solution, ready to use 28 C 12 weeks use only a clear solution

2025 C max. 2 weeks use only a clear solution

Stopping solution 28 C until expiry date

 

 

 

E. TEST PROCEDURE

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 37C and TMB-Chromogen / Substrate solution 10 to 20 min. at room temperature (2225 C). Adaptation of incubation periods to specific internal laboratory needs is possible, according to GLP they should be validated.

 

E.1 TEST PROCEDURE (summary)

 

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 37C

 

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. 2025 C)

 

PIPETTING OF THE STOPPING SOLUTION (0.100 ml)

PHOTOMETRIC MEASUREMENT at 450nm (Ref:630 nm)

 

TESTVALIDATION / EVALUATION

 

 

F. VALIDATION OF THE TEST, CORRECTIVE MEASURES

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.

 

G. EVALUATION

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.

 

H. INTERPRETATION OF RESULTS

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