BioShield 2019nCoV Total Ig, C1348C1396, is a semiquantitative ELISA test for the determination of IgA, IgM and IgG against SARSCoV2 in human serum or plasma specimens. The ELISA kit contains all reagents required for the immunoassay method. The ELISA test is adequate for 4896 definitions (controls are included). A spectrophotometer for microtiter ELISA plate is required
VERSION 2021-04-30/rev.05 C1348/C1396 Semi-quantitative ELISA Test | for the determination of Total Immunoglobulins (IgA, IgG and IgM) against SARS-CoV-2 in human serum or plasma specimens VERSION 2021-04-30/rev.05 VERSION 2021-04-30/rev.05 www.prognosis-biotech.com This ELISA kit is manufactured by ProGnosis Biotech S.A and complies with the specifications on the Standard EN ISO 13485:2016 Use only the current version of Product Data Sheet enclosed with the kit Bio-Shield 2019-nCoV Total Ig, C1348/C1396, is a semi-quantitative ELISA test for the determination of IgA, IgM and IgG against SARS-CoV-2 in human serum or plasma specimens The ELISA kit contains all reagents required for the immunoassay method The ELISA test is adequate for 48/96 definitions (controls are included) A spectrophotometer for microtiter ELISA plate is required Samples: Human serum or plasma specimens For in vitro diagnostic use only For a medical diagnosis, the serological test result should always be interpreted together with the clinical symptoms of the patient and other result Negative results not rule out SARS-CoV-2 infection Test should only be conducted by a medical personnel This test has not been reviewed by the FDA Results from antibody testing should not be used to diagnose or exclude SARS-CoV -2 infection or to inform infection status Positive results may be due to past or present infection with non-SARS-CoV-2 coronavirus strains, such as coronavirus HKU1, NL63, OC43, or 229E Not for the screening of donated blood Test time (incubation time after specimens and reagents preparation): 120 Shelf life: 12 months Storage: 2-8oC VERSION 2021-04-30/rev.05 Description Bio-Shield 2019-nCoV Total Ig is a semi-quantitative ELISA test for the determination of IgA, IgM and IgG against the Severe Acute Respiratory Syndrome CoronaVirus (SARS-CoV-2) in human serum or plasma specimens Intended Use The Bio-Shield 2019-nCoV Total Ig is an in vitro immunoassay, assisting in the diagnosis of patients with symptoms suggestive of SARS-CoV-2 infection At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity In combination with the clinical picture, results of RT-PCR and CT-Scan, Bio-Shield 2019-nCoV Total Ig could be used as an aid in the diagnosis of COVID-19 disease This immunoassay is a method for the determination of SARS-CoV-2 exposure levels, giving valuable information about the individuals’ immune status However, the interpretation of the results should not be used as the unique base of the SARS-CoV-2 infection status diagnosis All antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized Individuals may have detectable virus present for several weeks following seroconversion The sensitivity of Bio-Shield 2019-nCoV Total Ig early after infection is unknown Negative results not preclude acute SARS-CoV-2 infection If acute infection is suspected, direct testing for SARS-CoV-2 is necessary False positive results for Bio-Shield 2019-nCoV Total Ig may occur due to cross-reactivity from preexisting antibodies or other possible causes General Information Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses with a nucleocapsid of helical symmetry and are composed of several proteins including the Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) proteins The S protein is very immunogenic with the Receptor Binding Domain (RBD) being the target of many neutralizing antibodies The 2019 Novel Coronavirus, formerly known as 2019-nCoV and now known as SARS-CoV-2, emerged in the Chinese province of Hubei (Wuhan) in December 2019 and has been declared a pandemic on 11 March 2020 This outbreak has spread rapidly, with millions of reported cases and thousands of deaths worldwide Comparisons of the genetic sequences of this virus have shown similarities to SARS-CoV and bat coronaviruses The 2019nCoV disease (COVID-19) is a respiratory disease caused by infection with the SARS-CoV-2 virus and the incubation period of COVID-19 is generally days, with a minimum of and a maximum of 14 days Fever, cough, shortness of breath or difficulties in breathing, pain in the muscle and tiredness are the most common signs and symptoms of the virus In severe cases, the infection can further lead to pneumonia, severe acute respiratory syndrome, kidney failure and Antibody responses against SARS-CoV-2: Graph of positive rates of virus-specific IgG and IgM versus days after symptom onset in 363 serum samples from 262 patients (Long et al., 2020) (Published 29 April 2020) VERSION 2021-04-30/rev.05 death Currently, there is no medication or vaccine available against infection with this new virus Antibodies are produced by the immune system after an average of days after infection In general, Immunoglobulins M and A are the first immunoglobulins to be produced and IgG is the most abundantly found immunoglobulin that can be detected in the body for a long period Within 19 days after symptom onset, all patients tested are positive for IgG against SARS-CoV-2 Seroconversion for IgG and IgM occurs simultaneously or sequentially Both IgG and IgM titers reach a plateau within days after seroconversion Serological testing may be helpful for the diagnosis of suspected patients with negative RT–PCR results (determining the number of cases of COVID-19) and for the identification of asymptomatic infections2 Principle of the Method The immunoassay is based on the enzyme-linked immunosorbent assay principles The wells of the microtiter strips are coated with a mixture of different recombinant epitopes of SARS-CoV-2 S protein, including the RBD3 Diluted serum or plasma specimens, Positive controls, Cut-off controls and Negative controls are added into the wells of the microtiter plate Then, the antibody isotypes against SARSCoV-2 in specimens or controls (if present) bind to the coated recombinant S protein epitopes Any unbound immunoglobulin is removed with a washing step A detection solution with the same mixture of recombinant epitopes of SARS-CoV-2 S protein, conjugated to HRP, is added and a double antigen sandwich system is formed The detection reagent that has not reacted will be removed by a washing step A chromogen substrate is added to the wells resulting in the progressive development of a blue colored complex with the detection reagent The color development is then stopped by the addition of acid turning the resultant final product yellow The measurement is made photometrically at 450 nm and the intensity of the produced colored complex is proportional to antibodies present in the specimen The presence of total antibodies against SARS-CoV-2 S protein in an individual specimen is determined by comparing the optical density of the specimen to the optical density of the Cut-off control Reagents Provided Bio-Shield 2019-nCoV Total Ig ELISA kit contains sufficient reagents and materials for 48/96 measurements (including control tests) Reagents (Store at 2-8ºC) Quantity for 48 wells Quantity for 96 wells State Vial cap color 2019-nCoV Total Ig SingleBreak Strip Plate 48 wells 96 wells Ready to use (precoated) - Dilution Microwells 48 wells 96 wells Ready to use (red color) - Assay Diluent plastic vial (50 mL) plastic vials (50 mL) Ready to use White Negative Control plastic vial (0.3 mL) plastic vial (0.3 mL) Ready to use White Cut-off Control plastic vial (0.5 mL) plastic vial (0.5 mL) Ready to use White Positive Control plastic vial (0.3 mL) plastic vial (0.3 mL) Ready to use White 2019-nCoV Total Ig Detection Solution plastic vial (6 mL) plastic vial (12 mL) Ready to use Green Wash Buffer plastic vial (50 mL) plastic vial (50 mL) 20X Concentrate (dilute in distilled water) White TMB Substrate plastic vial (12 mL) plastic vial (12 mL) Ready to use Brown Stop Solution plastic vial (12 mL) plastic vial (12 mL) Ready to use White NOTE: Negative, Cut-off and Positive control not contain human serum Cut-off and Positive control contain chimeric recombinant antibodies against SARS-CoV-2 VERSION 2021-04-30/rev.05 Materials required but not provided Centrifuge, vortex mixer and microtiter plate reader fitted with 450 nm filter Adjustable single channel micropipettes of 10, 100 and 1000 μL with disposable tips (a repetitive pipette of 100-200 μL is preferable for the steps of Assay Diluent, Detection Solution, TMB and Stop Solution) and a multi-channel micropipette of 50 - 300 μL with disposable tips and reservoir Distilled or deionized water, a graduated cylinder of 1000 mL capacity, disposable plastic tubes and absorbent paper Protective goggles, gloves and container for biohazardous waste Storage Instructions Store kit reagents between and 8°C Do not freeze any components provided Reseal immediately the unused strips of the microtiter plate in the bag together with the desiccant bag provided and store at 2-8°C for month After use, all remaining reagents should be returned to 2-8°C where they also remain stable for month Expiry of the kit and reagents is stated on the labels respectively and no quality guarantee is accepted after the expiration date The expiry of the kit components can only be guaranteed if the components are stored properly as well as if the reagent is not contaminated by the first handling, in case of repeated use of one component Because of the colorless TMB Substrate light sensitivity, avoid exposure to direct light Safety and Precautions for use 8.1 Health and safety precautions Avoid any skin contact with Controls, Stop Solution (H3PO4 15%) and TMB Substrate (toxic) In case of contact, wash thoroughly with water Use gloves, protective clothing and eye/face protection and handle appropriately with the requisite Good Laboratory Practices The product must only be used by qualified personnel, familiar with the potential hazards, in a clinical or research laboratory Dispose of all specimens and materials used to perform the test as though they contain an infectious agent Laboratory, chemical or biohazardous wastes must be handled and discarded in accordance with the local, regional and national regulations Human source material spills should be also treated as potentially infectious Spills not containing acid should be immediately decontaminated, including the spill area, materials and any contaminated surfaces, with an appropriate chemical disinfectant (commonly 1:10 dilution of household bleach or 70-80% Ethanol) and should be wiped dry In accordance with Article 1, Paragraph 2b of European Directive 98/79/EC, the use of in-vitro diagnostic medical devices is envisaged by the manufacturer to ensure the suitability, performance, and safety of these products Consequently, the testing procedure, information, precautions, and warnings in the instructions for use must be followed rigorously No changes to the test procedure are permitted, nor is any use in combination with other products not approved by the manufacturer The user is solely responsible for any such changes The manufacturer is not responsible for false results nor incidents arising as a result of these The manufacturer is not responsible for any results obtained by visual analysis of patient samples Use a clean container to prepare the wash buffer and all residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper Never insert absorbent paper into the well Read the absorbance within 60 minutes after completion of the assay All reagents should be warmed in room temperature before use and covered when not in use Use a clean disposable plastic pipette tip for each reagent, to avoid cross-contamination When pipetting reagents, maintain a consistent order of addition from well-to-well This will ensure equal incubation times for all wells Do not interchange individual reagents between kits of different lot numbers 8.2 Precautions related to the procedure VERSION 2021-04-30/rev.05 Indication of spoilage of kit reagents The bluish coloration of the TMB substrate before the ELISA test Visible microbial contamination of reagents DO NOT USE the kit if the packaging of components is damaged, if there is an expired reagent or if the desiccant bag is absent inside microplate bag 10 Specimens and reagents preparation 10.1 Wash Buffer 1X preparation Dilute the 20X solution concentrate 20 fold with distilled water to give a 1X working solution In the case of the occurrence of crystals in the Wash Buffer, the warming of the crystals by gentle dismantling (using hands) is needed Pour the entire content of the solution concentrate (50 mL) into a clean 1000 mL graduated cylinder, rinse the vial with distilled or deionized water, pour the content again into the cylinder and fill to a final volume of 1000 mL with distilled or deionized water Mix gently to avoid foaming, transferring the final solution from cylinder to a clean bottle and back two times The clean bottle with 1X Wash Buffer working solution can be left out of the refrigerator during the method procedure and subsequently be stored 2-8°C for month 10.2 Serum and Plasma specimens Collection Blood specimens should be collected aseptically using venipuncture techniques by qualified personnel The Correct performance of specimen collection and storage is crucial for the test result Keep tubes sealed at all times The use of sterile or aseptic techniques will preserve the integrity of the specimens Serum: Use a serum separator tube (SST) and allow specimens to clot for hours at room temperature or overnight at 4oC Centrifuge the specimen for 10min at 4000xg at room temperature Remove serum and assay immediately or aliquot and store specimens at –20oC or –80oC Plasma: Collect plasma using Heparin, EDTA or Citrate as an anticoagulant Centrifuge for 15 minutes at 4000xg within 30 minutes of collection Assay immediately or aliquot and store specimens at –20oC or –80oC NOTE: Serum or plasma specimens can be subjected to a maximum of freezing/ thawing cycle Do not heat the specimens Avoid highly lipemic, icteric or hemolytic specimens because they could affect the results of the assay Specimens with visible microbial contamination should not be used, as they may affect the performance of the test After centrifugation, serum or plasma can be stored at 28oC if the test is performed within days 11 Method Procedure 11.1 Assay Design: Determine the number of microwell strips needed for the assay (Specimens, Positive Control x1, Cut-off Control x3 and Negative Control x1) Considering that each specimen and control can be tested in single, create a layout NOTE 1: If the number of wells is more than 32 (four strips), a repetitive pipette or multichannel pipette is necessary, for the Detection Solution, TMB Substrate and Stop Solution NOTE 2: The reliability of the test can be improved when duplicate values are used for each specimen VERSION 2021-04-30/rev.05 A POS B CUT OFF C CUT OFF D CUT OFF E NEG 10 11 12 F G H Example plate layout 11.2 Bring all reagents to room temperature (19 - 24°C) before use Remove the positive, Cut-off and Negative Control and place the appropriate number of Dilution Microwell (red) in a microwell holder for each Control or specimen to be tested Place an equal number of S protein Coated Microtiter Wells in another microwell holder Immediately reseal the unused strips of the microtiter plate in the bag together with the desiccant bag provided The specimens should be stored in a cool place 11.3 Add 200 μL of Assay Diluent to each Dilution Well 11.4 Using a new pipette tip for each, add 20 μL of each Control (Positive Control x1, Cut-off Control x3 and Negative Control x1) and serum or plasma specimens (see Chapter 10.2) to appropriate Dilution Well containing the Assay Diluent Mix by priming pipetting at least times NOTE: Use only sufficient volume of specimen as indicated in the procedure Failure to so may cause low sensitivity of the assay 11.5 Using a multichannel pipette, transfer 100μl of contents from each Dilution Microwell to a corresponding 2019-nCoV Total Ig Single-Break Strip Plate and incubate at room temperature for 60 minutes 11.6 When the countdown is over wash the plate as follows: Empty the liquid from each well (preferably in an appropriate container) and tap the holder of microwells upside down strongly (four times in a row) on an absorbent paper to ensure the complete removal of liquid from the wells Dispense 300μl of Wash Buffer 1X (see 10.1) into each well with a multichannel micropipette using the proper reagent reservoir and shake the plate manually for a few seconds Repeat this process for another four times (total times) CAUTION: It is important to not allow microwells dry between working steps 11.7 Aspirate the liquid as described above and add 100μl of Detection Solution to each well (pour 1ml per wells in a reservoir) Shake the plate manually for 30 seconds and incubate at room temperature for 45 minutes 11.8 Wash the plate as the wash step 11.6 11.9 Aspirate the liquid as described above and add 100μl of TMB Substrate to each well (pour 1ml per wells in a reservoir) Shake the plate manually for a few seconds and incubate in the dark at room temperature for 15 minutes 11.10 Add 100μl of the Stop Solution to each well (pour 1ml per wells in a reservoir) Mix gently by shaking again the plate manually 11.11 Measure the absorbance at 450nm Read the absorbance value of each well (within 60 minutes after the step 11.10) on a spectrophotometer using 450nm as the primary wavelength and VERSION 2021-04-30/rev.05 12 Data Analysis 12.1 Specification criteria Calculate the mean of measured Optical Density (OD) for the Cut-off Control (Cut-off Control ODMEAN) Specification criteria ODMEAN Cut-off Control 0,09 < Cut-off Control ODMEAN < 0,6 OD Positive Control / ODMEAN Cut-off Control OD Ratio ≥ 1,5 OD Negative Control / ODMEAN Cut-off Control OD Ratio ≤ 0,6 12.2 Interpretation of results Specimens results are expressed by Ratio using the following formula: Specimen Ratio = Specimen OD / Cut-off Control ODMEAN Interpretation of results Specimen Ratio < 0,8 Negative Result 0,8 ≤ Specimen Ratio < Equivocal Result Specimen Ratio ≥ Positive Result Specimen Ratio less than 0,8 is considered to be «negative» for the presence of anti-SARS-CoV-2 antibodies Specimen Ratio between 0,8 and 1,0 is considered to be «equivocal» for the presence of anti-SARS-CoV- antibodies It should be retested in duplicate before final interpretation In case of repeated equivocal results, another specimen should be collected and tested a few days later Specimen Ratio equal or more than 1,0 is considered to be «positive» for the presence of anti-SARS CoV- antibodies 13 Limitations of the immunoassay A single test result should not be the sole basis for the clinical diagnosis of SARS-CoV-2 Follow-up and supplemental testing as well as other clinical and laboratory data should be taken under consideration The detection of immunoglobulins against SARS-CoV-2 is dependent on the analyte concentration in the specimen A negative or non-reactive result can occur if the quantity of anti-SARS-CoV-2 antibodies present in the specimen is below the LOD of the assay During the acute infection phase and/or for immunosuppressed patients, anti-SARS-CoV-2 antibodies might not be detectable while the individual is infected by SARS-CoV-2 Thus, a negative result is not evidence for the absence of COVID19 infection The detection of antibodies against SARS-CoV-2 in serum or plasma is also linked to the frequency of the tests performed on the patients In order to increase the sensitivity and the earliness of the test positivity, regular monitoring of patients suspected to be infected by SARS-CoV-2 should be performed For a medical diagnosis, the serological test result should always be interpreted together with the clinical symptoms of the patient and other results, e.g those of the direct pathogen detection Negative results not rule out SARS-CoV-2 infection, particularly in those who have been in contact with the virus Specimens collected at the begging of infection or by some immunosuppressed individuals may not have detectable levels of antibodies In cases, at the early stage of infection, it is recommended to obtain a second specimen between 14 and 21 days to be tested in parallel with the original specimen, in order to determine a seroconversion VERSION 2021-04-30/rev.05 14 Immunoassay Performance 14.1 Precision 14.1.1 Intra-assay Three positive specimens and one negative specimen were assayed 42 times in the same run Specimen N Mean Ratio CV% Negative 42 0,33 6,8 Positive A 42 5,7 4,5 Positive B 42 17,1 3,9 Positive C 42 38,4 3,3 14.1.2 Inter-assay The same specimens were assayed in separate runs over 14 days Specimen N Mean Ratio CV% Negative 0,32 9,8 Positive A 5,8 4,2 Positive B 17,3 4,1 Positive C 38,9 3,5 14.2 Cross-reactivity Cross-reactivity has been evaluated by testing SARS-CoV-2 seronegative specimens from patients with antibodies to other coronaviruses or other pathogens A total of 68 specimens from 11 different categories were tested The number of samples tested for each category is listed below Pathogen Tested sample number False positive sample Influenza A virus Influenza B virus Hepatitis C virus Hepatitis B virus Hemophilus influenzae Alpha coronavirus 229E Alpha coronavirus NL63 Beta coronavirus OC43 Beta coronavirus HKU1 Antinuclear antibodies Respiratory syncytial virus 14.3 Hook effect In order to investigate the possibility of a hook effect, a positive specimen with high titer of Total immunoglobulins was serially diluted and was tested with Bio-Shield 2019-nCoV Total Ig assay No negative results were observed on the non diluted specimen A decrease of signal with the test of diluted specimens was observed 10 VERSION 2021-04-30/rev.05 14.4 Internal validation characteristics The internal validation of the Bio-Shield 2019-nCoV Total Ig was assessed during an evaluation of specimens obtained from a general asymptomatic population of pre-epidemic individuals (blood donors, hospitalized patients) and of specimens from patients with clinical symptoms of coronavirus COVID-19, which were tested and found positive with RT-PCR assay 14.4.1 Diagnostic Specificity A total of 669 specimens from Greece (blood donors or hospitalized asymptomatic patients), collected prior to the outbreak of the COVID-19 pandemic, were tested The specificity was 99.85%, 668/669 (CI: 99.16% to 99.97%) 14.4.2 Diagnostic Sensitivity An internal longitudinal study was performed on 168 patients from Greece with clinical symptoms of COVID-19 and with a PCR positive result Specimens per patient were collected from to 58 days post onset of clinical symptoms The sensitivity was 98.21%, 165/168 (CI: 94.87% to 99.63%) Days between onset of symptoms and sample collection Positive Specimens Nonreactive specimens Total % ≤ days 40 43 93 8-14 days 38 38 100 15-21 days 29 29 100 22-28 days 32 32 100 ≥ 29 days 26 26 100 Total 165 168 98.21 14.5 Clinical performance characteristics The clinical performance of the Bio-Shield 2019-nCoV Total Ig was conducted at AHEPA General University Hospital of Thessaloniki (referral hospital for SARS-CoV-2 in Greece) Specimens obtained from a general asymptomatic population of pre-epidemic individuals (blood donors, hospitalized patients) were used and patients with clinical symptoms of coronavirus COVID-19 tested positive with RT-PCR assay 14.5.1 Clinical Diagnostic Specificity A total of 118 specimens (blood donors or hospitalized asymptomatic patients), collected prior to the outbreak of the COVID-19 pandemic, was tested The specificity was 100%, 118/118 (CI: 96.92% to 100%) 14.5.2 Clinical Diagnostic Sensitivity An longitudinal study was performed on 125 patients with clinical symptoms of COVID-19 and with a PCR positive result Specimens per patient were collected from to 58 days post onset of clinical symptoms The sensitivity was 98.40%, 123/125 (CI: 89.85% to 98.22%) Days between onset of symptoms and sample collection Specimens Nonreactive specimens Total % ≤ days 19 21 90.47 - 14 days 24 24 100 15 - 21 days 30 30 100 22 - 28 days 23 23 100 29 - 50 days 27 27 100 Total 123 125 98.40 11 VERSION 2021-04-30/rev.05 15 Method Summary Total procedure time (after specimens and reagents preparation): 120 Mix 200 μL of the Assay Diluent with 20 μL of controls and specimens in the Dilution Microwells Transfer 100 μL from each well of the Dilution Microwells into the S-protein Coated Microwells Incubate 60 at room temperature Wash five times Add 100 μL of Detection Solution Incubate 45 at room temperature Wash five times Add 100 μL of TMB Substrate Let the color develop for 15 in the dark at room temperature Add 100 μL Stop Solution Read Absorbance at 450 nm 12 VERSION 2021-04-30/rev.05 16 References Long, Q., Liu, B., Deng, H et al Antibody responses to SARS-CoV-2 in patients with COVID19 Nat Med (2020) https://doi.org/10.1038/s41591-020-0897-1 Berry, J D., e t al Neutralizing epitopes of the SARS-CoV S-protein cluster independent of 446 repertoire, antigen structure or mAb technology MAbs 2, 53-66 (2010) Tai, W., He, L., Zhang, X et al Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine Cell Mol Immunol (2020) https://doi.org/10.1038/s41423-020-0400-4 13 VERSION 2021-04-30/rev.05 8°C In Vitro Diagnostics Medical Device n 2°C Storage Conditions Content sufficient for tests See Instruction for Use Catalog number Keep away from sunlight Manufacturer Negative Control Positive control 14 VERSION 2021-04-30/rev.05 15 VERSION 2021-04-30/rev.05 www.prognosis-biotech.com e: info@prognosis-biotech.com t: +30 2410 623922 | f: +30 700 700 6262 16