DRG EIA-3446 Adenovirus IgG ELISA Instruction Manual

Instructions for Use
Adenovirus IgG ELISA

INTRODUCTION

1.1 Intended Use
The DRG Adenovirus IgG Enzyme Immunoassay Kit provides materials for the qualitative and semiquantitative determination of IgG-class antibodies to Adenovirus in serum.
This assay is intended for in vitro diagnostic use only.

1.2 Summary and Explanation
Adenoviruses are double-stranded DNA viruses lacking an envelope, of about 60-90 nm. The capsid contains 252 capsomeres and shows icosahedral symmetry. The capsomeres consist of hexons, pentons, and fibers (so named after their configuration) which are responsible for the induction of group- and type- specific antibodies. In humans, 34 immunologically distinct types (serotypes) are recognized.
Adenoviruses were first isolated from adenoid tissue and have a certain affinity for lymph glands, where they may remain latent for years. They also invade the respiratory tract, the gastrointestinal tract, and the conjunctiva. Adenovirus infections are widely distributed and common with most infections occurring in childhood. The contagious disease normally is acute and self- limited, but infections may be prolonged and asymptomatic, possibly remaining latent for a very long time. Winter and Spring are the seasons for the highest rates of general adenovirus infections, independent of geographic locations and climatic conditions. Epidemics may occur in over-crowded populations, for example ARD in military groups, PCF in swimming pools, and EKC in medical facilities.

PRINCIPLE OF THE TEST

The DRG Adenovirus IgG ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) Microtiter wells as a solid phase are coated with inactivated Adenovirus Grade 2 antigen (strain Adenoid 6). Diluted patient specimens and ready-for-use controls are pipetted into these wells. During incubation Adenovirusspecific antibodies of positive specimens 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 Adenovirus-specific IgG antibody in the patient specimen. Absorbance 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 (21 °C – 26 °C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.

• Never pipette by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

• Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.

• Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens 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.

KIT COMPONENTS

4.1 Contents of the Kit

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with Adenovirus antigen.
   (incl. 1 strip holder and 1 cover foil)

2. Sample Diluent *, 1 vial, 100 mL, ready to use, colored yellow; pH 7.2 ± 0.2.

3. Pos. Control *, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.

4. Neg. Control *, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.

5. Cut-off Control *, 1 vial, 2.0 mL, ready to use; colored yellow, black cap.

6. Enzyme Conjugate *, 1 vial, 20 mL, ready to use, colored red, antibody to human IgG conjugated to horseradish peroxidase.

7. Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).

8. 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.

9. Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 6.5 + 0.1 see „Preparation of Reagents“.

• contain non-mercury preservative

4.1.1 Equipment and material required but not provided

• A microtiter plate calibrated reader (450/620nm ±10 nm) (e.g. the DRG Instruments Microtiter Plate Reader)
• Calibrated variable precision micropipettes
• Incubator 37 °C
• Manual or automatic equipment for rinsing wells
• Vortex tube mixer
• Deionised or (freshly) distilled water
• Timer
• Absorbent paper

4.2 Storage and stability of the Kit

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 two months if stored as described above.

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

Wash Solution
Dilute Wash Solution 1+19 (e.g. 10 mL + 190 mL) with fresh and germ free redistilled water. This diluted wash solution has a pH value of 7.2 ± 0.2.
Consumption: ~ 5 mL per determination.
Crystals in the solution disappear by warming up to 37 °C in a water bath. Be sure that the crystals are completely dissolved before use.
The diluted Wash Solution is stable for 4 weeks at 2 °C to 8 °C.

4.4 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 Material Safety Data Sheets (see chapter 13 of this data sheet).
4.5 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.

SPECIMEN COLLECTION AND PREPARATION

Serum can be used in this assay.
Do not use hemolytic, icteric or lipemic specimens.

5.1 Specimen Collection Serum:
Collect blood by venipuncture (e.g. Sestet Monolete 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.

5.2 Specimen Storage
Specimens should be capped and may be stored for up to 3 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time should be frozen only once at –20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

5.3 Specimen Dilution
Prior to assaying, dilute each patient specimen 1+1000 with Sample Diluent in a two-step dilution:

Step 1 (D1): 5 µL sample + 500 µL Sample Diluent ( = 1+100). Mix thoroughly!
Step 2 (D2):25 µL D1 + 250 µL Sample Diluent (= 1+1000). Mix thoroughly!

D2 is the final dilution of the sample.
Please note: Controls are ready for use and must not be diluted!

ASSAY PROCEDURE

6.1 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
• Absorbance 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 without splashing accurately to the bottom of wells.
• During 37°C incubation cover microtiter strips with foil to avoid evaporation.

6.2 Assay Procedure

Prior to commencing the assay, dilute Wash Solution, prepare patient samples as described in point 5.3, mix well before pipette and establish carefully the distribution and identification plan supplied in the kit for all specimens and controls.

1. 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 substrate blank,
   ---|---|---
   1 well| (e.g. B1)| for the Neg. Control,
   2 wells| (e.g. C1+D1)| for the Cut-off Control
   1 well| (e.g. E1)| for the Pos. Control.

It is left to the user to determine controls and patient samples in duplicate.

2. Dispense
   100 µL of Neg. Control into well B1
   100 µL of Cut-off Control into wells C1 and D1
   100 µL of Pos. Control into well E1 and
   100 µL of each d i l u t e d sample with new disposable tips into appropriate wells.
   Leave well A1 for substrate blank!

3. Cover wells with foil supplied in the kit. Incubate for 60 minutes at 37 °C.

4. 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!

5. Dispense 100 µL Enzyme Conjugate into each well, except A1.

6. Incubate for 30 minutes at room temperature (20 °C to 25 °C).
   Do not expose to direct sun light!

7. 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.

8. Add 100 µL of Substrate Solution into all wells.

9. Incubate for exactly 15 minutes at room temperature (20 °C to 25 °C) in the dark.

10. 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!

11. Read the optical density at 450/620 nm with a microtiter plate reader within 30 minutes after adding the Stop Solution.

6.3 Measurement
Adjust the ELISA microplate or microstrip reader to zero using the substrate blank in well A1.
If – due to technical reasons – the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results!
Measure the absorbance of all wells at 450 nm and record the absorbance 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 absorbance values of all duplicates.

RESULTS

7.1 Validation of the Test Run
The test run may be considered valid provided the following criteria are met:
Substrate blank in A1: Absorbance value lower than 0.100
Neg. Control in B1: Absorbance value lower than 0.200
Cut-off Control in C1/D1 : Absorbance value between 0.350 – 0.900
Pos. Control in E1: Absorbance value between 0.650 – 3.000
7.2 Calculation
Mean absorbance value of Cut-off Control [CO] Calculate the mean absorbance value of the two (2) Cut-off Control determinations (e.g. in C1/D1).
Example: (0.59 + 0.61) ∕ 2 = 0.60 = CO

7.3 Interpretation

POSITIVE| Patient (mean) absorbance values more than 10 % above CO (Mean OD patient   > 1.1 x CO)
---|---
GREY ZONE| Patient (mean) absorbance values from 10 % above to 10 % below CO
repeat test 2 – 4 weeks later – with new patient samples (0.9 x CO    ≤   Mean OD patient     ≤   1.1 x CO)
Results in the second test again in the grey zone  Þ  NEGATIVE
NEGATIVE| Patient (mean) absorbance values more than 10 % below CO (Mean OD patient   < 0.9 x CO)

7.3.1 Results in DRG Units [DU]

Interpretation of Results

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

9.1 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 %.
9.2 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 %.

LIMITATIONS OF USE

Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values.
In immunocompromised patients and newborns serological data only have restricted value.

LEGAL ASPECTS

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.

11.2 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.

11.3 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 / LITERATURE

Donelli,G.,F.Superti,A.Tinari et al.: Viral childhood diarrhoe in Rome: a diagnostic and epidemiological study.
Microbiologica 16 (1993) 215-225
Hierholzer,J.C.: Adenovirus in the immunocompromised host.
Clin.Microbiol.Rev.5 (1992) 262-274
Schmitz,H.,R.Wiegand,W.Heinrich: Worlwide epidemiology of human adenovirus infectionsw.
Am.J.Epid.117 (1983) 455-466

SYMBOLS USED

| European Conformity
---|---
| Consult instructions for use
| In vitro diagnostic medical device
| Catalogue number
| Batch code
| Contains sufficient for tests
| Temperature limit
| Use-by date
| Manufacturer
| Distributor
| Date of manufacture
| Biological risks
| Caution
| Unique device Identifier *
| For research use only
Content| Content
Volume/No.| Volume / No
Microtiter wells| Microtiter wells
Enzyme Conjugate| Enzyme Conjugate
Substrate Solution| Substrate Solution
Stop Solution| Stop Solution
Zero Standard| Zero Standard
Standard| Standard
Control| Control
Pos. Control| Positive Control
Neg. Control| Negative Control
Cut-off Control| Cut-off Control
Wash Solution| Wash Solution
Sample Diluent| Sample Diluent
Conjugate Diluent| Conjugate Diluent

SHORT INSTRUCTIONS FOR USE

| All reagents and specimens must be allowed to come to room
temperature (18-25°C) before use.
---|---
| Leave well Al for substrate Blank.
Dispense 100 pl of Controls into appropriate wells.
| Dispense 100 pl of sample into selected wells.
(Please note special sample treatment, point 5.31)
| 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 pl per well).
| Strike the wells sharply on absorbent paper to remove residual droplets.
| Dispense 100 pl 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 pl per well).
| Strike the wells sharply on absorbent paper to remove residual droplets.
| Add 100 pl of Substrate Solution to each well.
| Incubate for 15 minutes at room temperature.
| Stop the reaction by adding 100 pl of Stop Solution to each well.
| Determine the absorbance of each well at 450 nm.

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

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081
Phone: 973-564-7555, Fax: 973-564-7556
Website: www.drg-international.com
E-mail: corp@drg-international.com

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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