DRG INTERNATIONAL EIA-3510 Rubella IgG ELISA Instruction Manual

DRG INTERNATIONAL EIA-3510 Rubella IgG ELISA

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

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

Summary and Explanation

Rubella is an enveloped RNA virus belonging to the togavirus. It has a spherical shape measuring about 50-70 nm in diameter. There appears to be only one antigenic type, and no cross-reactivity with alphavirus or other members of the togavirus group has been found.

Rubella viruses are pathogens of the respiratory tract and transmitted mainly by droplet infection. Rubella is a worldwide common contagious disease with mild constitutional symptoms and a generalized rash. In childhood, it is an inconsequential illness, but when it occurs during pregnancy, there is a significant risk of severe damage to the fetus. The risk of congenital rubella depends primarily on the month of pregnancy in which infection is acquired: overall, app. 16% of infants have major defects at birth following maternal rubella in the first 3 months of pregnancy. Congenital rubella infection may lead to a syndrome with single or multiple organ involvements, known as embryopathia rubeolosa. In some cases infection is inapparent but results in consequential damages as eye defects, deafness, growth retardation, and others. Naturally acquired immunity usually is long-lasting, but reinfection is possible due to decreasing levels of circulating antibodies. For immunization a vaccine containing live virus is used.
Rubella Infection may be identified by detection of virus by PCR (prenatal), Hemagglutination inhibition (HAI), Haemolysis-in-gel test (HiG), detection of antibodies by EIA, ELISA.

Measurement of antibodies in the serum is important for the determination of the immune status. Even a previous infection though rather overt may not yield a long-lasting immunity, but may result in an antibody titer too low to prevent reinfection. Especially the screening of adolescents and young women should be a mandatory routine in prenatal care.
Rubella Antibody detection:

six months

PRINCIPLE OF THE TEST

The DRG Rubella IgG ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA).
Microtiter wells as a solid phase are coated with inactivated K1S grade Rubella Virus antigen (strain HPV-77).

Diluted patient specimens and ready-for-use controls are pipetted into these wells. During incubation Rubella Virus-specific 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 Rubella Virus-specific IgG antibody in the patient specimen. 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 (21 °C to 25 °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.

REAGENTS

Reagents provided

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells;
   Wells coated with inactivated K1S grade Rubella Virus antigen (strain HPV-77).
   (incl. 1 cover foil)

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

3. Standard (Standard 1 – 3)*, 3 vials, ready to use;
   Concentrations: 10, 50, 100 IU/mL,
   The standards are calibrated against WHO International Standard Anti Rubella Immunglobulin, Human (RUBI-1-94) Standard 1, 2 mL, colored green, green cap;
   Standard 2, 2 mL, colored blue, blue cap;
   Standard 3, 2 mL, colored red, red cap.

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

5. Pos. Control*, 1 vial, 2.0 mL, ready to use; colored purple, black cap.

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

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.

Material required but not provided

• A microtiter plate calibrated 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 (20 °C to 25 °C) 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 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.

SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (EDTA, lithium heparin or citrate plasma) can be used in this assay.
In general, it should be avoided to use haemolytic, icteric or lipaemic specimens. For further information refer to chapter “Interfering Substances”

Specimen 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.

Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 4 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 15 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Specimen Dilution
Prior to assaying dilute each patient specimen 1+100 with Sample Diluent;
e.g. 10 µL of specimen + 1 mL of Sample Diluent mix well, let stand for 15 minutes mix well before use.
For patients with concentrations greater than Standard 3 a second 1:10 dilution of this 1+100 diluted patient sample should be performed;
e.g. 20 µL of first sample dilution + 180 µL Sample Diluent (mix well).
Please note: Standards and 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 without splashing accurately to the bottom of wells.
• During 37 °C 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 and establish carefully the distribution and identification plan supplied in the kit for all standards, 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 Neg. Control,
   4 wells (e.g. from B1 on) for the Standard 1 – 3
   1 well (e.g. F1) for the Pos. Control

2. Dispense

3. 100 μL of Neg. Control into well A1

4. 100 μL of Standard 1 into well B1 + C1

5. 100 μL of Standard 2 into well D1

6. 100 μL of Standard 3 into well E1

7. 100 μL of Pos. Control into well F1 and

8. 100 μL of each diluted sample with new disposable tips into appropriate wells.

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

10. 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!

11. Dispense 100 µL Enzyme Conjugate into each well.

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

13. 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.

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

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

16. 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!

17. 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.

CALCULATION OF 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 0.20
• Standard 1 (Cut-off) in B1 + C1:    OD value between 0.35 – 0.85
• Standard 2 in D1:                                OD value between 0.90 – 2.10
• Standard 3 in E1: OD value between 1.40 – 2.60
• Pos. Control in F1: OD value between 0.65 – 3.00

Calculation of quantitative Results

In order to obtain quantitative results in IU/mL plot the (mean) OD values of Neg. Control and Standards 1, 2, 3 on (linear/linear) graph paper in a system of coordinates against their corresponding concentrations (0, 10, 50 and 100 IU/mL) and draw a standard calibration curve (OD values on the vertical y-axis, concentrations on the horizontal x-axis).

Read results from this standard curve employing the (mean) OD values of each patient specimen and control.
All validated computer programs can be used, that provide the following function: 4 PL (4 Parameter Logistics).
DRG uses the DRG regression program for Windows (4 parameter Rodbard regression).
If other regression software is used, the obtained values have to be validated by the user.

NOTE : Values of additionally (1:10, in total 1:1000) diluted patient samples must be multiplied by the appropriate dilution factor in order to obtain correct results! (Dilution: 1:10 = Dilution factor: 10). (See chapter “5.3 Specimen Dilution”).

Interpretation of quantitative Results
The following values should be considered as a guideline:

• NEGATIVE                                                < 10 IU/mL
• GREY ZONE (equivocal):                          10 – 15 IU/mL
• POSITIVE:                                                  > 15 IU/mL

Important notes for the interpretation of results of the DRG Rubella IgG ELISA
If a Rubella infection is suspected during pregnancy it is required to determine the antibody state of the pregnant woman. In case that 65 IU/mL IgG is found in serum and higher, the woman is protected against the infection and there is no risk of an embryonic damage. Results found from 40 – 45 IU/mL are in a grey-zone.

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.

PERFORMANCE CHARACTERISTICS

1. Assay Dynamic Range
   The range of the assay is between 0.40 – 100 IU/mL.

2. Specificity of Antigen (Cross Reactivity)
   No cross reactivity was found for Measles, Mumps, Varicella zoster Virus, Epstein-Barr Virus (VCA), Parvovirus B19 and Dengue-2 Virus.

3. 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 zero standard (= negative control) and was found to be 0.40 IU/mL (OD450nm 0.030).

4. Diagnostic Specificity
   The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. (Detected by method comparison with NovaTec ELISA with three lots of DRG ELISA, 185 samples, therefrom 27 negative samples are assayed.)
   It is 100% for all three DRG production lots.

5. Diagnostic Sensitivity
   The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. (Detected by method comparison with NovaTec ELISA, 185 samples, therefrom 158 positive samples are assayed.)
   It is 100% for all three DRG production lots.

6. Method Comparison
   The DRG ELISA was compared with other Rubella IgG ELISA (NovaTec). 185 serum samples are assayed.n =| 185| Other ELISA
   ---|---|---
   pos.| neg.

DRG ELISA

| pos.| 158| 0
neg.| 0| 27
7. Reproducibility

1. Intra-assay
   The intra-assay (within-run) precision of the DRG Rubella IgG ELISA was determined by 20 x measurements of 12 serum samples covering the whole measuring range. Sample| Mean Conc. (IU/mL)| Intra-Assay CV (%)| n
   ---|---|---|---
   1| 8.0| 9.8| 20
   2| 0.4| 9.5| 20
   3| 5.0| 8.9| 20
   4| 10.7| 9.0| 20
   5| 21.2| 5.9| 20
   6| 11.1| 8.2| 20
   7| 27.8| 8.9| 20
   8| 46.8| 7.6| 20
   9| 43.9| 9.5| 20
   10| 93.7| 8.2| 20
   11| 140.1| 9.2| 20
   12| 157.7| 9.9| 20
2. Inter-assay
   The inter-assay variation of the DRG Rubella IgG ELISA was determined with 3 samples with 2 production kits in 10 independent runs with 2 replicates per run. Sample| Mean Conc. (IU/mL)| Inter-Assay CV (%)| n
   ---|---|---|---
   1| 12.8| 11.5| 40
   2| 18.2| 11.4| 40
   3| 22.1| 14.7| 40
3. Recovery
   Recovery of the DRG Rubella IgG ELISA was determined by adding increasing amounts of the analyte to three different sera containing different amounts of endogenous analyte. The percentage recoveries were determined by comparing expected and measured values of the samples. OD values / DRG Units should increase with addition of increasing analyte concentrations. | Sample 1| Sample 2| Sample 3
   ---|---|---|---
   Concentration [IU/mL]| 4.5| 4.8| 1.1
   Average Recovery| 99.7| 91.4| 98.5

Range of Recovery [%]

| from| 94.0| 87.7| 91.0
to| 110.6| 94.1| 106.5
4. Linearity
Three samples (serum) containing different amounts of analyte were serially diluted with sample diluent and assayed with the DRG ELISA. The percentage recovery was calculated by comparing the expected and measured values for the analyte. | Sample 1| Sample 2| Sample 3
---|---|---|---
Concentration [IU/mL]| 180.5| 100.2| 146.0
Average Recovery| 96.7| 104.6| 102.2

Range of Recovery [%]

| from| 85.3| 91.9| 91.2
to| 104.4| 115.0| 114.4
8. 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.

1.  **Interfering Substances**  

Haemoglobin (up to 4 mg/mL), Bilirubin (up to 0.25 mg/mL) and Triglyceride (up to 7.5 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.

SYMBOLS USED

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
IgG-RF Sorbent| Rheumatoid factor absorbent
Sample Diluent| Sample Diluent
Conjugate Diluent| Conjugate Diluent

SHORT INSTRUCTIONS FOR USE

References

• Analyticon® Biotechnologies GmbH

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