DRG International Zika Virus IgG Capture ELISA

Specifications:

• Product Name : Zika Virus IgG (capture) ELISA
• Model Number : EIA-5984
• Manufacturer: DRG International, Inc.
• Address : 841 Mountain Ave., Springfield, NJ 07081
• Phone : 973-564-7555
• Fax : 973-564-7556
• Website : www.drg-international.com
• Email : corp@drg-international.com
• Version : 4.0
• Date : 2023-08-15

Instructions for Use – Zika Virus IgG (capture) ELISA

Introduction

Zika Virus (ZIKV) is a single-stranded RNA virus of the Flaviviridae family (genus Flavivirus). It was first isolated in 1947 from a sentinel rhesus monkey during a yellow fever study in the Zika forest of Uganda. Since its discovery, ZIKV circulation has been detected in Africa and Asia where it has caused sporadic human infections. In 2007 its emergence on Yap Island, Micronesia was reported, marking transmission of Zika virus outside Africa and Asia. Since 2013, ZIKV has been reported from French Polynesia, New Caledonia, Cook Islands, Easter Island (Chile), Samoa and Vanuatu, and in early 2015 it spread initially to Brazil and subsequently to additional countries of the Americas. ZIKV is transmitted primarily through the bite of an infected Aedes species mosquito (A. aegypti and A. albopictus). However, there have been reports of less common transmission modes, such as blood transfusion, perinatal, and sexual contact. The incubation period of Zika virus disease is not known precisely, but is likely to be a few days. It is estimated that only one in five people infected with ZIKV develop signs or symptoms. Clinical manifestations of ZIKV infection are described as very similar to those of Dengue virus (DENV) and Chikungunya virus (CHIKV) infections, but usually milder. The most common clinical signs and symptoms are maculopapular rash, low-grade fever, arthralgia, myalgia, headache, and conjunctivitis. Less frequently reported are edema, sore throat, cough, vomiting, and hematospermia.

Intended Use

The Zika Virus IgG capture ELISA is intended for the qualitative determination of IgG class antibodies against Zika virus in human serum or plasma (citrate, heparin).

Principle of the Assay

The qualitative immunoenzymatic determination of specific IgG-class antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) capture technique. The intensity of this product is proportional to the amount of specific IgG antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA microtiter plate reader.

FAQ

• Q: What is the intended use of the Zika Virus IgG capture ELISA?
  • The Zika Virus IgG capture ELISA is intended for the qualitative determination of IgG class antibodies against Zika virus in human serum or plasma (citrate, heparin).
• Q: How is the assay principle based on the ELISA capture technique?
  • The assay principle is based on the immunoenzymatic determination of specific IgG-class antibodies using the ELISA capture technique. The intensity of the product is proportional to the amount of specific IgG antibodies in the sample.
• Q: What is the recommended reading method for the assay?
  • The absorbance at 450/620 nm should be read using an ELISA microtiter plate reader.

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

INTRODUCTION

• Zika Virus (ZIKV) is a single-stranded RNA virus of the Flaviviridae family (genus Flavivirus). It was first isolated in 1947 from a sentinel rhesus monkey during a yellow fever study in the Zika forest of Uganda.
  Since its discovery, ZIKV circulation has been detected in Africa and Asia where it has caused sporadic human infections. In 2007 its emergence on Yap Island, Micronesia was reported, marking transmission of Zika virus outside Africa and Asia. Since 2013, ZIKV has been reported from French Polynesia, New Caledonia, Cook Islands, Easter Island (Chile), Samoa and Vanuatu, and in early 2015 it spread initially to Brazil and subsequently to additional countries of the Americas.
  ZIKV is transmitted primarily through the bite of an infected Aedes species mosquito (A. aegypti and A. albopictus). However, there have been reports of less common transmission modes, such as blood transfusion, perinatal, and sexual contact.
  The incubation period of Zika virus disease is not known precisely, but is likely to be a few days.

• It is estimated that only one in five people infected with ZIKV develop signs or symptoms. Clinical manifestations of ZIKV infection are described as very similar to those of Dengue virus (DENV) and Chikungunya virus (CHIKV) infections, but usually milder.
  The most common clinical signs and symptoms are maculopapular rash, low grade fever, arthralgia, myalgia, headache and conjunctivitis. Less frequently reported are oedema, sore throat, cough, vomiting, and haematospermia.

• Human infections with ZIKV are usually mild and self-limiting and the symptoms usually resolve spontaneously after 3 – 7 days; arthralgia may persist for up to 1 month. In rare cases, after a Zika virus infection a Guillain-Barré syndrome (GBS), a disorder of the peripheral nerves, can probably occur. A correlation between a Zika virus infection in pregnancy and congenital brain malformations is now considered likely.

Species| Disease| Symptoms (e.g.)| Transmission route
---|---|---|---
Zika Virus (ZIKV)| Zika fever| Fever, headache, retro-orbital pain, conjunctivitis, maculopapular rash, myalgias, arthralgias| Primary mode of transmission via bite of Aedes

mosquitos

Infection or presence of pathogen may be identified by :

• Isolation in cell culture
• PCR
• Serology : Detection of antibodies by IF, ELISA Plaque Reduction Neutralization Test (PRNT)

INTENDED USE

The Zika Virus IgG capture ELISA is intended for the qualitative determination of IgG class antibodies against Zika virus in human serum or plasma (citrate, heparin).

PRINCIPLE OF THE ASSAY

The qualitative immunoenzymatic determination of specific IgG-class antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) capture technique.

Microtiter plates are coated with anti-human IgG-class antibodies to bind the corresponding antibodies of the sample. After washing the wells to remove all unbound sample material a horseradish peroxidase (HRP) labelled Zika virus antigen is added. This antigen-conjugate binds to the captured specific IgG antibodies. In a second washing step unbound conjugate is removed. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of specific IgG antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA microtiterplate reader.

MATERIALS

Reagents supplied

• Microtiterplate (IgG): 12 break-apart 8-well snap-off strips coated with anti-human IgG-class antibodies; in resealable aluminium foil.
• Sample Dilution Buffer : 1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1).
• Stop Solution : 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap.
• Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap.
• Conjugate : 1 bottle containing 15 mL of peroxidase labelled Zika virus antigen; coloured blue; ready to use; black cap; ≤ 0.02% (v/v) MIT.
• TMB Substrate Solution : 1 bottle containing 15 mL 3,3′,5,5′-tetramethylbenzidine (TMB), < 0.1 %; ready to use; yellow cap.
• Positive Control: 1 vial containing 2 mL control; coloured yellow; ready to use; red cap; ≤ 0.02% (v/v) MIT.
• Cut-off Control : 1 vial containing 3 mL control; coloured yellow; ready to use; green cap; ≤ 0.02% (v/v) MIT.
• Negative Control : 1 vial containing 2 mL control; coloured yellow; ready to use; blue cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1).

For hazard and precautionary statements see 12.1.

For potential hazardous substances please check the safety data sheet.

Materials supplied

• 1 Cover foil
• 1 Instruction for use (IFU)
• 1 Plate layout

Materials and Equipment needed

• ELISA microtiter plate reader, equipped for the measurement of absorbance at 450/620 nm
• Incubator 37 °C
• Manual or automatic equipment for rinsing microtiter plates
• Pipettes to deliver volumes between 10 and 1000 μL
• Vortex tube mixer
• Distilled water
• Disposable tubes

STABILITY AND STORAGE

Store the kit at 2 °C – 8 °C. The opened reagents are stable up to the expiry date stated on the label when stored at 2 °C – 8 °C.

REAGENT PREPARATION

It is very important to bring all reagents and samples to room temperature (20 °C – 25 °C) and mix them before starting the test run!

Microtiter plate

The break-apart snap-off strips are coated with anti-human IgG-class antibodies. Immediately after removal of the strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2 °C – 8 °C.

Washing Buffer (20x conc.)

Dilute Washing Buffer 1 + 19; e.g. 10 mL Washing Buffer + 190 mL distilled water. The diluted buffer is stable for 5 days at room temperature (20 °C – 25 °C). In case crystals appear in the concentrate, warm up the solution to 37 °C, e.g. in a water bath. Mix well before dilution.

TMB Substrate Solution

The reagent is ready to use and has to be stored at 2 °C – 8 °C, away from the light. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away.

SAMPLE COLLECTION AND PREPARATION

Use human serum or plasma (citrate, heparin) samples with this assay. If the assay is performed within 5 days after sample collection, the samples should be kept at 2 °C – 8 °C; otherwise they should be aliquoted and stored deep- frozen (-70 °C to -20 °C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. Heat inactivation of samples is not recommended.

Sample Dilution

Before assaying, all samples should be diluted 1+100 with Sample Dilution Buffer. Dispense 10 μL sample and 1 mL Sample Dilution Buffer into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.

ASSAY PROCEDURE
Please read the instruction for use carefully before performing the assay. Result reliability depends on strict adherence to the instruction for use as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend increasing the washing steps from three up to five and the volume of Washing Buffer from 300 μL to 350 μL to avoid washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and identification plan for all samples and standards/controls (duplicates recommended) should be carefully established on the plate layout supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. Perform all assay steps in the order given and without any delays.
A clean, disposable tip should be used for dispensing each standard/control and sample.

Adjust the incubator to 37 °C ± 1 °C.

1. Dispense 100 μL standards/controls and diluted samples into their respective wells. Leave well A1 for the Substrate Blank.
2. Cover wells with the foil supplied in the kit.
3. Incubate for 1 hour ± 5 min at 37 °C ± 1 °C.
4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300 μL of Washing Buffer. Avoid overflows from the reaction wells. The interval between washing and aspiration should be > 5 seconds. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is important! Insufficient washing results in poor precision and false results.
5. Dispense 100 μL Conjugate into all wells except for the Substrate Blank well A1.
6. Incubate for 30 min at 37 °C ± 1 °C. Do not expose to direct sunlight.
7. Repeat step 4.
8. Dispense 100 μL TMB Substrate Solution into all wells.
9. Incubate for exactly 15 min at room temperature (20 °C – 25 °C) in the dark. A blue colour occurs due to an enzymatic reaction.
10. Dispense 100 μL Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution, thereby a colour change from blue to yellow occurs.
11. Measure the absorbance at 450/620 nm within 30 min after the addition of the Stop Solution.

Measurement

Adjust the ELISA microtiterplate reader to zero using the Substrate Blank.

If – due to technical reasons – the ELISA microtiter plate reader cannot be adjusted to zero using the Substrate Blank, subtract its absorbance value 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 standard/control and sample in the plate layout.

RESULTS

Run Validation Criteria

In order for an assay run to be considered valid, these Instructions for Use have to be strictly followed and the following criteria must be met:

• Substrate Blank : Absorbance value < 0.100
• Negative Control : Absorbance value < Cut-off
• Cut-off Control : Absorbance value 0.150 – 1.300
• Positive Control : Absorbance value > Cut-off

If these criteria are not met, the test is not valid and must be repeated.

Calculation of Results

The Cut-off is the mean absorbance value of the Cut-off Control determinations.
Example : Absorbance value Cut-off Control 0.44 + absorbance value Cut- off control 0.42 = 0.86 ∕ 2 = 0.43 Cut-off = 0.43

Results in Units [DU]

Sample (mean) absorbance value × 10 = [DRG Units = DU] Cut-off
Example : 1.591 × 10 = 37 DU 0.43

Interpretation of Results

Positive

| ****

11 DU

| Antibodies against the pathogen are present.

There has been a contact with the antigen (pathogen resp. vaccine).

Equivocal

| ****

9 – 11 DU

| Antibodies against the pathogen could not be detected clearly.

It is recommended to repeat the test with a fresh sample in 2 to 4 weeks. If the result is equivocal again the sample is judged as negative.

Negative

| ****

< 9 DU

| The sample contains no antibodies against the pathogen.

A previous contact with the antigen (pathogen resp. vaccine) is unlikely.

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.

In immunocompromised patients and newborns serological data only have restricted value.

SPECIFIC PERFORMANCE CHARACTERISTICS

• The results refer to the groups of samples investigated; these are not guaranteed specifications.
• For further information about the specific performance characteristics please contact DRG.

Precision

intra-assay n Mean (E) CV (%)

1 24 0.422 6.7

2 24 1.062 2.1

3 24 0.988 2.6

Inter-assay n Mean (DU) CV (%)

1 12 32.60 6.2

2 12 22.57 5.7

3 12 23.84 5.9

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 99.6 % (95 % confidence ce interval: 97.92 % – 99.99 %).

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 98.1 % (95 % confidence interval: 89.93 % – 99.95 %).

Interferences

Three clinical samples exhibiting differing reactivities were tested for interference with each substance listed in the table below: a positive, a negative, and an equivocal sample. All samples exhibited a change of signal less than 15 % when tested with each potential interferant.

Cross Reactivity

Investigation of a sample panel with antibody activities to potentially cross- reacting parameters did not reveal significant evidence of false-positive results due to cross-reactions. However, in endemic areas, double infection as well as past infection with other flaviviruses should be considered.

LIMITATIONS OF THE PROCEDURE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

PRECAUTIONS AND WARNINGS

• The test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the test kits with analysers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples.
• Only for in-vitro diagnostic use.
• All materials of human or animal origin should be regarded and handled as potentially infectious.
• All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive.
• Do not interchange reagents or microtiterplates of different production lots.
• No reagents of other manufacturers should be used along with reagents of this test kit.
• Do not use reagents after expiry date stated on the label.
• Use only clean pipette tips, dispensers, and lab ware.
• Do not interchange screw caps of reagent vials to avoid cross-contamination.
• Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
• After first opening and subsequent storage check conjugate and standard/control vials for microbial contamination prior to further use.
• The ELISA is only designed for qualified personnel following the standards of good laboratory practice (GLP).
• For further internal quality control each laboratory should additionally use known samples.

Safety note for reagents containing hazardous substances

Reagents may contain CMIT/MIT (3:1) or MIT (refer to 4.1) Therefore, the following hazard and precautionary statements apply.

Warning

• H317 May cause an allergic skin reaction.
• P261 Avoid breathing spray
• P280 Wear protective gloves/ protective clothing.
• P302+P352 IF ON SKIN: Wash with plenty of soap and water.
• P333+P313 If skin irritation or rash occurs: Get medical advice/ attention.
• P362+P364 Take off contaminated and Wash it before reuse.

Further information can be found in the safety data sheet.

Disposal Considerations

Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is regulated through national and regional laws and regulations. Contact your local authorities or waste management companies which will give advice on how to dispose hazardous waste.

BIBLIOGRAPHY

1. Lanciotti, Robert S.; Kosoy, Olga L.; Laven, Janeen J.; Velez, Jason O.; Lambert, Amy J.; Johnson, Alison J. et al. (2008): Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. In Emerging infectious diseases 14 (8), pp. 1232–1239. DOI: 10.3201/eid1408.080287
2. Lazear, Helen M.; Diamond, Michael S. (2016): Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere. In Journal of virology 90 (10), pp. 4864–4875. DOI: 10.1128/JVI.00252-16.
3. Musso, Didier; Gubler, Duane J. (2016): Zika Virus. In Clinical Microbiology Reviews 29 (3), pp. 487–524. DOI: 10.1128/CMR.00072-15.
4. Petersen, Lyle R.; Jamieson, Denise J.; Powers, Ann M.; Honein, Margaret A. (2016): Zika Virus. In The New England Journal of Medicine 374 (16), pp. 1552–1563. DOI: 10.1056/NEJMra1602113.
5. Rasmussen, Sonja A.; Jamieson, Denise J.; Honein, Margaret A.; Petersen, Lyle R. (2016): Zika Virus and Birth Defects–Reviewing the Evidence for Causality. In The New England Journal of Medicine 374 (20), pp. 1981–1987. DOI: 10.1056/NEJMsr1604338.
6. Waggoner, Jesse J.; Pinsky, Benjamin A. (2016): Zika Virus: Diagnostics for an Emerging Pandemic Threat. In Journal of Clinical Microbiology 54 (4), pp. 860–867. DOI: 10.1128/JCM.00279-16.
7. Weaver, Scott C.; Reisen, William K. (2010): Present and future arboviral threats. In Antiviral research 85 (2), pp. 328–345. DOI: 10.1016/j.antiviral.2009.10.008.
8. Zammarchi, Lorenzo; Stella, Giulia; Mantella, Antonia; Bartolozzi, Dario; Tappe, Dennis; Gunther, Stephan et al. (2015): Zika virus infections imported to Italy: clinical, immunological and virological findings, and public health implications. In Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 63, pp. 32–35. DOI: 10.1016/j.jcv.2014.12.005.

Abbreviations

SCHEME OF THE ASSAY

Test Preparation

• Prepare reagents and samples as described.
• Establish the distribution and identification plan for all samples and standards/controls on the plate layout supplied in the kit.
• Select the required number of microtiter strips or wells and insert them into the holder.

Assay Procedure

| Substrate Blank (A1)| Negative Control| Cut-off Control| Positive Control| Sample (diluted 1+100)
---|---|---|---|---|---
Negative Control| –| 100 µL| –| –| –
Cut-off Control| –| –| 100 µL| –| –
Positive Control| –| –| –| 100 µL| –
Sample (diluted 1+100)| –| –| –| –| 100 µL
Cover wells with foil supplied in the kit

Incubate for 1 h at 37 °C ± 1 °C

Wash each well three times with 300 µL of Washing Buffer

Conjugate| –| 100 µL| 100 µL| 100 µL| 100 µL
Incubate for 30 min at 37 °C

Do not expose to direct sunlight

Wash each well three times with 300 µL of Washing Buffer

TMB Substrate Solution| 100 µL| 100 µL| 100 µL| 100 µL| 100 µL
Incubate for exactly 15 min at room temperature (20 °C – 25 °C) in the dark
Stop Solution| 100 µL| 100 µL| 100 µL| 100 µL| 100 µL
Photometric measurement at 450 nm (reference wavelength: 620 nm)

SYMBOLS USED

Contacts

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

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

References

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

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