DS-EIA-ANTI-SARS-CoV-2-SPECTRUM

“DS-EIA-ANTI-SARS-CoV-2-SPECTRUM” kit is an enzyme immunoassay for the detection of total antibodies (including IgM, IgA, IgG) to the nucleocapsid and spike protein of SARS-CoV-2 virus separately in human serum or plasma.
The kit may be used to evaluate the immune response to recent or prior coronavirus infection, to determine the acute infection in combination with other assays and studies, to confirm positive or grey zone results, discrimination of false positive results.
According to Interim Guidelines for COVID-19 Antibody Testing, CDC from 01.08.2020, one of approach to improve positive predictive value is to employ an orthogonal testing algorithm in which persons who initially test positive are tested with a second test. Effective orthogonal algorithms are generally based on testing a patient sample with two tests, each with unique design characteristics (e.g., antigens or formats). Thus total antibodies detection to the spike and nucleocapsid protein separately allows minimizing the false positive results of serological testing.
This kit is intended for clinical diagnostic use by trained laboratory professionals only and will not be sold to the general public. All the reagents are for professional in vitro diagnostic use only.

INTRODUCTION
The 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-SPECTRUM” is a “sandwich” immunoassay for the detection of IgG, IgM, IgA antibodies to SARS-CoV-2 (COVID-19). Recombinant antigens with the SARS-CoV-2 sequences are immobilized onto the surface of the polystyrene plate: six strips of the plate are coated with the antigens representing the sequence of nucleocapsid, and the other six strips – with the sequences of receptor-binding domain of the surface S-glycoprotein. If specific IgG, IgM, IgA antibodies to SARS-CoV-2 are present in the samples, an immune antigen-antibody complex is formed that is detected by the conjugate – mixture of the recombinant antigens to SARS-CoV-2, labelled with horseradish peroxidase.
After the plate is washed to remove the unbound components and the Substrate Mixture (TMB + Substrate Buffer) is added to the plate wells, the solution develops a blue colour. The reaction is stopped by the addition of Stopping Reagent changing the colour from blue to yellow. Colour intensity is proportional to the quantity of antibodies to SARS-CoV-2 in the sample. Absorbance is measured spectrophotometrically at 450/620-680 nm or 450 nm.

CONTENTS OF THE “DS-EIA-ANTI-SARS-CoV-2-SPECTRUM”
Contents of the reagent kit.

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.
- Disposable pipette tips.
- Thermoshaker at (37.0 ± 1.0) °C, 500 rpm.
- Automatic microplate washer.
- Microplate reader equipped with 450 nm or with 450 and 620-680 nm filters.
- Laboratory clock.

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. Sampling devices containing liquid anticoagulants have a dilution effect resulting in lower values for individual patient samples. For citrated plasma (1 part citrate solution + 9 parts blood), the dilution effect must be taken into account.
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. Do not heat the samples.
One freezing/defrosting cycle of the samples is permissible.
Samples with expressed bacterial growing, hemolysis, hyperlipidemia should not be tested.