DRG B19 IgG ELISA Parvovirus Instructions

: June 5, 2024
: DRG

DRG B19 IgG ELISA Parvovirus Instructions

INTENDED USE

The DRG Parvovirus B19 IgG (Recombinant) Enzyme Immunoassay Kit provides materials for the qualitative and semiquantitative determination of IgG-class antibodies to Parvovirus B19 in human serum and plasma (EDTA, lithium heparin or citrate plasma).
This assay is intended for in vitro diagnostic use only.

Summary and Explanation

Parvoviruses are cubic single-stranded DNA viruses of about 18-32 nm lacking an envelope. Parvovirus B19 infects only humans, and since there are no cross- reactivities between animal parvoviruses and B19, transmission between pets and humans is not possible. Parvovirus B19 is the causative agent of Erythema infectiosum, the so-called “fifth disease”, a mild rash illness that occurs most commonly in children. Infected persons are contagious during the early part of the illness before the rash appears so in adults the rate of epidemia amounts to about 60 %. About 20 % of adults and children who are infected with parvovirus B19 do not develop any symptoms. Persons infected with the virus, however, do develop lasting immunity that protects them against infection in the future. Parvovirus B19 infection may cause a serious illness in persons with sickle-cell disease or similar types of chronic anemia as well as in persons who have problems with their immune system (people with leukemia or cancer, who are born with immune deficiencies, who have received an organ transplant, or who have HIV infection). Occasionally (less than 5 % of all pregnant women infected with parvovirus B19) serious complications may develop during pregnancy: risk of Morbus haemolyticus fetalis.

PRINCIPLE OF THE TEST

The Parvovirus B19 IgG ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) Microtiter wells as a solid phase are coated with recombinant Parvovirus B19 antigen (VP1-s and VP2-s proteins). Diluted patient samples and ready-for-use controls are pipetted into these wells. During incubation Parvovirus B19- specific antibodies of positive samples and controls are bound to the immobilized antigens. After a washing step to remove unbound sample and control material horseradish peroxidase conjugated anti-human IgG antibodies are dispensed into the wells. During a second incubation this anti IgG conjugate binds specifically to IgG antibodies resulting in the formation of enzyme-linked immune complexes.
After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid.
The intensity of this color is directly proportional to the amount of Parvovirus B19-specific IgG antibody in the patient sample. Optical density at 450 nm is read using an ELISA microtiter plate reader.

WARNINGS AND PRECAUTIONS

This kit is for in vitro diagnostic use only. For professional use only.
Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
Avoid contact with Stop Solution containing 0.2 mol/L H2SO4. It may cause skin irritation and burns.
TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (20 °C to 25 °C) before starting the test. Temperature will affect the optical density readings of the assay. However, values for the patient samples will not be affected.
Never pipette by mouth and avoid contact of reagents and samples with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where samples or kit reagents are handled.
Wear disposable latex gloves when handling samples and reagents. Microbial contamination of reagents or samples may give false results.
Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guideline or regulation.
For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG.

REAGENTS

Reagents provided

Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with recombinant Parvovirus B19 antigen (VP1-s and VP2-s protein). (incl. 1 cover foil)
Sample Diluent *, 1 vial, 100 mL, ready to use, colored yellow; pH 7.2 ± 0.2.
Pos. Control *, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.
Neg. Control *, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.
Cut-off Control *, 1 vial, 2.0 mL, ready to use; colored yellow, black cap.
Enzyme Conjugate *, 1 vial, 20 mL, ready to use, colored red, antibody to human IgG conjugated to horseradish peroxidase.
Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).
Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/L H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.
Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 6.5 ± 0.1 see „Preparation of Reagents“.

contain non-mercury preservative

Material required but not provided

A calibrated microtiter plate reader (450 nm/620 nm ± 10 nm)
Calibrated variable precision micropipettes
Incubator 37 °C
Manual or automatic equipment for rinsing wells
Vortex tube mixer
Freshly distilled water
Timer
Absorbent paper

Storage Conditions

When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 8 weeks if stored as described above.

Reagent Preparation

Allow all reagents and required number of strips to reach room temperature prior to use.

Wash Solution
Add fresh and germ-free distilled water to the 20X concentrated Wash Solution.
Dilute the complete content of the vial 1 + 19 (30 mL Wash Solution + 570 mL distilled water) to a final volume of 600 mL.
If crystals have formed in the wash solution concentrate, ensure that they are completely transferred into the solution.
This diluted wash solution has a pH value of 7.2 ± 0.2.

Consumption: ~ 5 mL per determination.
The diluted Wash Solution is stable for 1 week at 2 °C to 8 °C.

Disposal of the Kit

The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Safety Data Sheets.

Damaged Test Kits

In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations.

SAMPLE COLLECTION AND PREPARATION

Serum or plasma (EDTA, lithium heparin or citrate plasma) can be used in this assay.

Please note: Samples containing sodium azide should not be used in the assay.
In general, it should be avoided to use haemolytic, icteric or lipaemic samples. For further information refer to chapter “Interfering Substances”.

Sample Collection

Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

Plasma:
Whole blood should be collected into centrifuge tubes containing anti coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

Sample Storage

Samples should be capped and may be stored for up to 5 days at 2 °C to 8 °C prior to assaying. Samples held for a longer time (up to 18 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Sample Dilution

Prior to assaying dilute each patient sample 1+100 with Sample Diluent;
e.g. 10 µL of sample + 1 mL of Sample Diluent, mix well, let stand for 15 minutes, mix well again.

Please note: Controls are ready for use and must not be diluted!

ASSAY PROCEDURE

General Remarks

It is very important to bring all reagents, samples and controls to room temperature before starting the test run!
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination
Optical density is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.
Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate withoutsplashing accurately to the bottom of wells.
During incubation cover microtiter strips with foil to avoid evaporation.

Test Procedure

Prior to commencing the assay, dilute Wash Solution, prepare patient samples as described in point 5.3, mix well before pipetting. Carefully establish the plate layout sheet supplied in the kit for all samples and controls.

Select the required number of microtiter strips or wells and insert them into the holder.
Please allocate at least:
1 well (e.g. A1) for the Neg. Control,
2 wells (e.g. B1 + C1) for the Cut-off Control and
1 well (e.g. D1) for the Pos. Control.
It is left to the user to determine controls and patient samples in duplicate.
Dispense
100 µL of Neg. Control into well A1
100 µL of Cut-off Control into wells B1 + C1
100 µL of Pos. Control into well D1 and
100 µL of each diluted sample with new disposable tips into appropriate wells.
Cover wells with foil supplied in the kit. Incubate for 60 minutes at 37 °C.
Briskly shake out the contents of the wells.
Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.
Important note: The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!
Dispense 100 µL Enzyme Conjugate into each well.
Incubate for 30 minutes at room temperature (20 °C to 25 °C). Do not expose to direct sun light!
Briskly shake out the contents of the wells.
Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.
Add 100 µL of Substrate Solution into all wells.
Incubate for exactly 15 minutes at room temperature (20 °C to 25 °C) in the dark.
Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.
Any blue color developed during the incubation turns into yellow.
Note: Highly positive patient samples can cause dark precipitates of the chromogen!
Read the optical density at 450/620 nm with a microtiter plate reader within 30 minutes after adding the Stop Solution.

Measurement

Measure the optical density (OD) of all wells at 450 nm and record the OD values for each control and patient sample in the distribution and identification plan.
Dual wavelength reading using 620 nm as reference wavelength is recommended.
Where applicable calculate the mean OD values of all duplicates.

RESULTS

Validation of the Test Run

The test run may be considered valid provided the following criteria are met:

Neg. Control in A1: OD value lower than 0.200
Cut-off Control in B1/C1: OD value between 0.350 – 0.850
Pos. Control in D1: OD value between 0.650 – 3.000

The OD value of the Pos. Control should be higher than the OD value of the Cut-off Control. (OD Pos. Control > OD Cut-off Control).

Calculation

Mean OD value of Cut-off Control [CO]
Calculate the mean OD value of the two (2) Cut-off Control determinations (e.g. in B1/C1).
Example: (0.40 + 0.45) ∕ 2 = 0.425 = CO

Interpretation

POSITIVE: Patient (mean) OD values more than 20 % above CO (Mean ODpatient > 1.2 × CO)
GREY ZONE: Patient (mean) OD values from 20 % above to 10 % below CO repeat test 2 – 4 weeks later – with new patient samples (0.90 × CO ≤ Mean ODpatient ≤ 1.2 × CO)
Results in the second test again in the grey zone  NEGATIV
NEGATIVE: Patient (mean) OD values more than 10 % below CO (Mean ODpatient < 0.90 × CO)

Results in DRG Units [DU]

Patient (mean) OD value × 10/CO= [DRG Units = DU] Example: 1.210 × 10/0.47 = 25 DU

Interpretation of Results

Cut-off value: 10 DU
Grey zone: 9 – 12 DU
Negative: < 9 DU
Positive: > 12 DU

QUALITY CONTROL

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. After checking the above mentioned items without finding any error contact your distributor or DRG directly.

ASSAY CHARACTERISTICS

Analytical Sensitivity

The analytical sensitivity of the DRG ELISA was calculated by adding 2 standard deviations to the mean OD of 20 replicate analyses of the negative control and was found to be 0.59 DU/mL (OD 450 nm = 0.034).

Diagnostic Specificity

The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. It is 100 %.

Diagnostic Sensitivity

The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. It is 100 %.

Method Comparison

The DRG ELISA was compared with the Mikrogen Parvovirus B19 IgG. 74 serum samples are assayed.

n = 74	Reference Assay
pos.	neg.
DRG ELISA	pos.
neg.	0

Agreement: 100 %

Reproducibility

Intra-assay

The intra-assay (within-run) precision of the DRG ELISA was determined by 20x measurements of 12 samples covering the measuring range of the ELISA.

Sample	n	Mean OD	Intra-Assay CV ( %)
1	20	0.03	9.7
2	20	0.26	5.7
3	20	0.07	6.0
4	20	0.99	8.8
5	20	0.76	5.9
6	20	0.71	9.9
7	20	1.23	6.8
8	20	1.37	3.7
9	20	1.25	3.4
10	20	1.54	3.9
11	20	2.00	3.6
12	20	2.03	1.7

Inter-assay
The inter-assay variation of the DRG ELISA was determined in 20 independent runs with 2 replicates per run.

Sample	n	Mean OD	Intra-Assay CV ( %)
1	20	2.26	7.2
2	20	1.75	9.9
3	20	2.32	9.3

LIMITATIONS OF USE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the OD values.
In immunocompromised patients and newborns serological data only have restricted value.
10.1 Interfering Substances
Hemoglobin (up to 4 mg/mL), bilirubin (up to 0.5 mg/mL) and triglyceride (up to 30 mg/mL) have no influence on the
assay results.

LEGAL ASPECTS

Reliability of Results

The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test. The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

Therapeutic Consequences

Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient. Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis should take into consideration clinical history, symptomatology as well as serological data. Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutic consequences.

Liability

Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

REFERENCES

Tino F. Schwarz and Gundula Jäger: A recombinant immunoblot and Elisa for detection of acute Parvovirus B 19 infection Zbl. Bakt. 1994, 280, 526-533
Maria Söderlund, Caroline S. Brown, Willy J. M. Spaan, Lea Hedman and Klaus Hedman, Epitope type-specific IgG response to capsid proteins VP1 and VP2 of human Parvovirus B19. The Journal of infectious Deseases 1995, 72, 1431-1436
P. Cassinotti, Human Parvovirus B19 infections and their diagnosis Alpe Adria Microbiologiy Journal 1995, 4, 235-246
M. Schleuning, Parvovirus B19 Infektionen Deutsches Ärzteblatt 93, Heft 43, (Oktober 1996), B2182-B2185
A von Poblotzki , A Gigler, B Lang, H Wolf, S Modrow: Antibodies to Parvovirus B 19 NS-1 protein in infected individuals. J. Gen. Virology 1995, 76, 519-527

SYMBOLS USED

Symbol

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| European Conformity
| Consult instructions for use
| In vitro diagnostic medical device

SHORT INSTRUCTIONS FOR USE

| All reagents and samples must be allowed to come to room temperature (20 °C – 25 °C) before use.
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| Dispense 100 µL of Controls into appropriate wells.
| Dispense 100 µL of sample into selected wells.
(Please note special sample treatment, point 5.3!)
| Cover wells with foil.
Incubate for 60 minutes at 37 °C.
| Briskly shake out the contents of the wells.
| Rinse the wells 5 times with diluted Wash Solution (300 µL per well).
| Strike the wells sharply on absorbent paper to remove residual droplets.
| Dispense 100 µL of Enzyme-Conjugate into each well.
| Incubate for 30 minutes at room temperature.
| Briskly shake out the contents of the wells.
| Rinse the wells 5 times with diluted Wash Solution (300 µL per well).
| Strike the wells sharply on absorbent paper to remove residual droplets.
| Add 100 µL of Substrate Solution to each well.
| Incubate for 15 minutes at room temperature.
| Stop the reaction by adding 100 µL of Stop Solution to each well.
| Determine the optical density of each well at 450/620 nm.

DRG Instruments GmbH, Germany
Frauenbergstraße 18, 35039 Marburg
Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50
Website: www.drg-diagnostics.de
E-mail: [email protected]