DS-EIA-ANTI-SARS-CoV-2-G (S)

Description

Description

DS-ЕIA-ANTI-SARS-CoV-2-G (S): ENZYME IMMUNOASSAY FOR THE DETECTION OF IgG ANTIBODIES TO THE SARS-CoV-2 SPIKE PROTEIN

“DS-EIA-ANTI-SARS-CoV-2-G (S)” kit is an enzyme immunoassay for the detection of IgG antibodies to the SARS-CoV-2 spike protein that causes coronavirus infection COVID-19, in human serum or plasma.
The kit may be used to evaluate the immune response to recent or prior coronavirus infection (COVID-19), to determine the acute infection only in combination with other researches, and for determination of specific IgG antibodies titer.
The results of this or any other diagnostic assay should be used and interpreted only in the context of the overall clinical picture.
Serological assay can be applied to collect epidemiological data.
This kit is for diagnostic use by a trained laboratory professional (in laboratory setting) and will not be sold to the general public. All the reagents are for professional in vitro diagnostic use only.
Target populations for the detection of IgG antibodies to the SARS-CoV-2 spike protein are patients with active SARS-CoV-2 infections or convalescent patients.

INTRODUCTION
The disease COVID-19 is caused by coronavirus SARS-CoV-2 (2019-nCoV). This virus is the seventh coronavirus that has been proven to infect humans. It is the third coronavirus that has emerged in the past 2 decades, causing multinational outbreaks and carrying substantial morbidity and mortality. There are no specific clinical features of COVID-19 and symptoms are similar to those of other severe acute respiratory infections, such as MERS and SARS [1].
Four structural proteins are essential for the life cycle of COVID-19. Homotrimers of S proteins make up the spikes on the surface of virus particles and it is the key for the viral attachment to host receptor. The M protein has three transmembrane domains and it shapes the virions. The E protein plays a role in virus assembly and release. The N protein contains two domains, both of them can bind virus RNA [2].

PRINCIPLE OF THE TEST
The kit “DS-EIA-ANTI-SARS-CoV-2-G (S)” is an indirect two-step immunoassay for the detection of IgG antibodies to the SARS-CoV-2 spike protein. Recombinant antigens with the SARS-CoV-2 sequence are immobilized onto the surface of the polystyrene plate. If IgG antibodies to SARS-CoV-2 are present in the sample, an immune antigen-antibody complex is formed (step 1) that is detected by the conjugate-human IgG antibodies labelled with horseradish peroxidase (step 2).
After the Substrate Mixture (TMB + Substrate Buffer) is added to the plate wells, the solution develops a blue color. The reaction is stopped by the addition of Stopping Reagent changing the color from blue to yellow. Color intensity is proportional to the quantity of antibodies to SARS-CoV-2 in the tested sample. Absorbance is measured spectrophotometrically at 450 nm or 450/620 nm.

CONTENTS OF THE “DS-EIA-ANTI-SARS-CoV-2-G (S)”

Contents of the reagent kit

LABEL	NATURE OF THE REAGENTS	PRESENTATION
CoV-2 Coated Strips	Polystyrene stripped 96-well plate (breakable wells) coated with SARS-CoV-2 spike protein. Store at 2-8 °C until expiration date.	1
Conjugate (concentrated 21-fold)	Anti-human IgG antibodies, peroxidase-labeled in glycerol-based solution with addition of phosphate-saline solution (4%) and bovine serum albumin (1.7%). Transparent or slightly opalescent liquid, colorless or pale yellow colored. Preserving agents: 0.04% ProClin 300, 0.0009% gentamicin sulfate. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 0.6 ml
Conjugate Diluent	Phosphate-saline solution with addition of urea (6%) and bovine serum albumin (4%). Transparent or slightly opalescent yellow liquid. Preserving agent: 0.10% ProClin 300, 0.10% phenol. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 12.5 ml
Positive Control, Inactivated	Heat inactivated human serum positive for IgG antibodies to SARS-CoV-2, negative for HIV-1 p24 antigen, anti-HIV-1,2, HBsAg and anti-HCV with addition of bovine serum albumin (2%). Transparent or slightly opalescent liquid, red colored. Preserving agent: 0.10 % ProClin 300, 0.10% sodium azide, 0.10% phenol. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 1.2 ml
Negative Control, Inactivated	Heat inactivated human serum negative for HIV-1 p24 antigen, anti-HIV-1,2, HBsAg and anti-HCV, anti-SARS-CoV-2 with addition of urea (4.55%). Transparent or slightly opalescent liquid, green colored. Preserving agent: 0.04 % thimerosal, 0.01% gentamicin sulfate, 0.02% sodium azide. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 2.0 ml
Sample Diluent	Phosphate-saline solution with addition of urea (2.91%). Transparent or slightly opalescent liquid, pink colored. Sediment may form. Preserving agent: 0.01 % ProClin 300, 0.097% sodium azide. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 12.5 ml
Preliminary Sample Diluent	Phosphate-saline solution. Transparent or slightly opalescent liquid, violet-blue colored. Preserving agent: 0.09% sodium azide. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 12.5 ml
Washing Solution (concentrated 25-fold)	Phosphate-saline solution (pH 7.4-7.7). Contains Tween-20 (2.75%). Transparent or slightly opalescent liquid, colorless or pale yellow, sediment may form that dissolves at 35-39 °C and shaking. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 50.0 ml
Stopping Reagent	Sulfuric acid solution (H2SO4) 0.2M. Transparent colorless liquid. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 25.0 ml
Substrate Buffer	citric acid (0.64%) solution (pH 4.1-4.3), containing H2O2; colorless liquid; preservatives: ProClin 300 0.04 %	1 × 25 ml
Stopping reagent 0.2M H2SO4	Citric acid (0.64%) solution, pH 4.1-4.3, containing H2O2 (0.008%). Transparent colorless liquid. Preserving agent: 0.04 % ProClin 300. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 25.0 ml
TMB (concentrated 11-fold)	Solution containing 3,3’,5,5’-Tetramethylbenzidine (TMB) (0.37%) and dimethyl sulfoxide (DMSO) (85.66%). Transparent colorless liquid, coloration is possible. Store at 2-8 °C until expiration date in a tightly sealed vial.	1 vial x 2.5 ml
Plate for preliminary dilution of sera	Polystyrene plate with transparent wells.	1 plate
Protective films for EIA plates		2
Disposable tips		16
Disposable plastic dishes for liquid reagents		2
Polyethylene bag with a Zip-Lock		1

PRECAUTIONS
The reliability of the results depends on correct implementation of the following requirements:
- The temperature in the lab should be 18-24 °C.
- Inspect the contents of the box: check the vials and labels integrity. In case of label loss or labels/vials damage, vials should be disposed and kit cannot be used.
- Do not use expired reagents.
- Do not mix reagents from different lots within a given test run.
- Carefully reconstitute the reagents avoiding any contamination.
- Do not carry out the test in the presence of reactive vapors (acid, alkaline, aldehyde vapors) or dust that could alter the enzyme activity of the conjugates.
- Use glassware thoroughly washed and rinsed with deionized water or preferably, disposable material.
- Do not allow the microplate to dry between the end of the washing operation and the reagent distribution.
- The enzyme reaction is very sensitive to metal ions. Consequently, do not allow any metal element to come into contact with the various conjugate or substrate solutions.
- Use a new distribution tip for each sample.
- Do not reuse protective films for EIA plates.
- Each well of CoV-2 Coated Strips is for single use.
- Well washing is a critical step in this procedure: respect the recommended number of washing cycles and make sure that all wells are completely filled and then completely emptied. Incorrect washing may lead to inaccurate results.
- Never use the same container to distribute conjugate and other solutions
- Check the pipettes and other equipment for accuracy and correct operation.
- Do not change the assay procedure.
- Use high quality water.
- Avoid exposure of the reagents to excessive heat or sunlight during storage and incubation.
- Once the assay has been started, all subsequent steps should be performed without interruption.

HEALTH AND SAFETY INSTRUCTIONS
- All reagents included in the kit are intended for “in vitro diagnostic use”.
- Human origin material used in the preparation of the Control samples has been tested and found non-reactive for hepatitis B surface antigen (HBsAg), antigen p24 HIV-1, antibodies to hepatitis C virus and antibodies to human immunodeficiency virus (HIV-1 and HIV-2).
- Because no known test method can offer complete assurance that infectious agents are absent, handle reagents and patients samples as if capable of transmitting infections disease.

- Do not eat, drink, smoke, or apply cosmetics where immunodiagnostic materials are being handled.
- Any equipment directly in contact with specimens and reagents as well as washing solutions should be considered as contaminated products and treated as such.
- Wear lab coats and disposable gloves when handling reagents and samples and thoroughly wash your hands after handling them.
- Avoid spilling samples or solutions containing samples.
- Avoid any contact of the Substrate Buffer, the TMB and the Stopping Reagent with the skin and mucosa.
- Provide adequate ventilation.
- All materials contacted with specimens or reagents, including liquid and solid wastes, should be inactivated by validated procedures (autoclaving or chemical treatment) and disposed in accordance with applicable local law regulations.

MATERIALS AND EQUIPMENT REQUIRED BUT NOT PROVIDED WITH THE TEST
- Distilled or deionized water.
- Automatic or semiautomatic, adjustable or preset single-channel and multi-channel pipettes with a changeable volume for a set of liquids (to measure and dispense 10 μl, 90 μl, 100 μl, 150 μl, 1 ml).
- Graduated cylinders of 10 ml, 100 ml, 1000 ml.
- Disposable pipette tips.
- Microplate incubator at (37.0 ± 1.0) °C.
- Automatic microplate washer with possibility of 4 times washing with not less than 350 μl washing solution.
- Microplate reader equipped with 450 nm or with 450 and 620-680 nm filters.

- Laboratory clock.
- Open type automated analyzer with 450 nm or with 450 and 620-680 nm filters (for automated procedure).

COLLECTION AND HANDLING OF SPECIMENS
Blood samples should be collected according to the current practices. Use native (undiluted) human serum or plasma (heparin, EDTA, citrate) for the assay. Separate the serum or plasma from the clot or red cells as soon as possible to avoid any hemolysis. Extensive hemolysis may affect test performance. Specimens with observable particulate matter should be clarified by centrifugation prior testing. Suspended fibrin particles or aggregates may yield falsely positive results. Heat inactivated human blood serum/plasma samples (30 min at 56 °C) may be used for assay. Separated serum/plasma should be stored/transported for no more 48 hours at 2 to 8 °C. If samples are to be stored/transported for a longer period of time, they must be frozen at or below -20 °C. Two cycles freezing/defrosting is permissible.
Samples with expressed bacterial growing, hemolysis, hyperlipidemia should not be tested.

PREPARATION OF THE REAGENTS
1. Ready for use reagents:
- SARS-CoV-2 Coated Strips. Each 12-strips plate (breakable wells) is wrapped in a sealed foil-lined bag. Open the bag and remove the plate. Select the number of strips/wells required for the assay. Place the unused strips/wells back into the foil-lined bag; reseal the foil-lined bag in a Zip-Lock plastic bag. Do not remove desiccant.
- Positive Сontrol
- Negative Сontrol
- Sample Diluent
- Preliminary Sample Diluent
- Stopping Reagent
2. Reagents to prepare
- Working Washing Solution. Thoroughly mix the contents of the bottle with concentrated Washing Solution (concentrated 25-fold). Dilute the required volume of concentrated Washing Solution with the corresponding volume of distilled or deionized water prior to use (See Table 2). Mix the solution thoroughly.
Working Solution of Conjugate. Thoroughly mix the contents of the vial with concentrated Conjugate. For the working solution of Conjugate preparation, dilute the required volume of Conjugate (concentrated 21-fold) with the required volume of Conjugate Diluent (See Table 2) and mix thoroughly avoiding foaming.
- Substrate Mixture. Dilute the required volume of TMB (concentrated 11-fold) with the corresponding volume of Substrate Buffer (See Table 2). Mix thoroughly.
It is possible to prepare Substrate Mixture directly in the vial with Substrate Buffer. For this purpose, pipette the required volume of TMB (2.5 ml) in to the vial with Substrate Buffer (25.0 ml) and mix thoroughly.
Substrate Mixture should be colorless!