MicroELISA

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Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative detection of IgG antibodies to SARS-CoV-2in human serum or plasma. It is intended for evaluating the immune response of patients suspected to be infected by SARS-CoV-2, for seroepidemiologic studies and as an aid in the diagnosis of Coronavirus disease 2019 (COVID-19). Positive results can help to qualifypeople to donate blood that can be used to manufacture convalescent plasma for those who are seriously ill from COVID-19. The serological test will be used for the monitoring of the immunological response upon vaccination (when available). For “in vitro” diagnostic use only.

SARS-CoV-2 is a new strain of Coronavirus that was first identified during an outbreak in Wuhan, China, in December 2019. Coronaviruses are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). Coronaviruses are named for their crown-like appearance when imaged. The large, type I transmembrane spike glycoprotein accounts for this notable feature. It is a heavily-glycosylated, cell-surface protein which is thought to mediate viral entry into susceptible cells. This spike glycoprotein, called ‘S’, is trimeric in structure. In addition to the S protein, there are three other structural base proteins: the envelope, membrane, and nucleocapsid. The S protein has two distinct functional domains, termed S1 and S2, both of which are necessary for a Coronavirus to successfully enter a cell.SARS-CoV-2 is the ethiological agent of COVID-19 respiratory disease. Common signs of infection include respiratory symptoms, fever, cough, shortness of breath, breathing difficulties, anosmia and ageusia. In severe cases ,infection can cause pneumonia, severe acute respiratory syndrome (SARS), kidney failure and death.

The spike and nucleocapsid proteins are major immunogenic components of CoV-2 and are produced in abundant quantities during acute infection.

Microplates are coated with SARS-CoV-2 Nucleocapsid protein and Spike protein recombinant antigens. The solid phase is first incubated with the diluted sample and IgG to SARS-CoV-2 are captured, if present, by the antigens. After washing out all the other components of the sample, in the 2^nd incubation bound anti SARS-CoV-2 IgG are detected by the addition of polyclonal specific anti hIgG antibodies, labelled with peroxidase (HRP). The enzyme captured on the solid phase, acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of anti SARS-CoV-2 IgG antibodies present in the sample. Calibrators, calibrated against the NIBSC serum code 20/130 (Version 1, Dated 30/04/2020), makes possible a quantitative determination of the IgG antibody in the patient.

Each kit contains sufficient reagents to perform 96 tests (code 081138).

1. Calibrated Micropipettes (1000 µL, 100 µL, 10 µL) and disposable plastic tips.
2. EIA grade water (double distilled or deionised, charcoal treated to remove oxidizing chemicals used as disinfectants).
3. Timer with 60 minute range or higher.
4. Absorbent paper tissues.
5. Calibrated ELISA microplate thermostatic incubator (dry or wet), set at 37°C (± 1.0°C tolerance).
6. Calibrated ELISA microwell reader with 450nm (reading) and possibly with 620-630nm (blanking) filters.
7. Calibrated ELISA microplate washer.
8. Vortex or similar mixing tools.

1.   Analitical Specificity

Potentially Cross-Reacting Antibodies

The SARS-CoV-2 IgG assay was evaluated for potentially cross-reacting antibodies to other viruses that  cause symptoms similar to SARS-CoV-2 infection. A total of 35 specimens from 7 different categories were tested.

34 specimens were negative and 1 specimen was Indeterminate by the SARS-CoV-2 IgG assay.

The data are summarized in the following table.

1. Diagnostic Specificity

Donors

Testing was performed on 206 European unselected blood donors collected prior to Covid 19 pandemic (before September 2019):

3. Analytical Sensitivity (Limit of Detection)

The limit of detection of the assay has been calculated by means of the anti SARS-CoV-2 Ab  NIBSC code 20/130.

The limit of detection is  defined as the concentration of SARS-CoV-2 IgG  equivalent to the mean absorbance of Calibrator A plus five standard deviations based on 20 replicates.

The assay shows a limit of detection of 0,03 arbU/mL.

4. Diagnostic Sensitivity

Nr 67 specimens were collected at different time from 42 patients who tested positive for SARS-CoV-2 by a polimerasi chain reaction (PCR) method.

Positive Percent Agreement (TP) and 95% confident limit (Cl) between the U-SARS-CoV-2 IgG Assay and PCR were calculated.

5. Precision

It has been tested in 4 replicates each in 5 individual runs in order to calculate and compare the within and the total assay CV%.

Results are reported as follows:

Intra-Lot Results: