Clinical Validation of a Simple and Fast Immunochromatographic Assay for Qualitative Determination of Specific ImmunoGLObulin IgG/IgM Antibodies to 2019- nCoV in Whole bLood, Serum or Plasma Specimen
Conditions: COVID
Sponsor: European Institute of Oncology

Performance Study Plan Versione 1.1_03.04.2020 
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Clinical validation of a Simple and Fast Immunochromatographic assay for 
qualitative determination of specific ImmunoGLObulin IgG/IgM Antibodies to 
2019- nCoV in whole bLood, serum or plasma specimen. 
 
Code: I GLOBAL  
SPONSOR: Istituto Europeo di Oncologia 
 
Coordinating Investigator 
 
Prof. Giuseppe Curigliano 
Istituto Europeo di Oncologia 
Via Ripamonti, 435 
 20141 Milano 
 
 
Participating Sites:  
 
Prof. Daniele Generali  
Azienda Socio-Sanitaria Territoriale di Cremona 
Viale Concordia, 1 
26100 Cremona 
 
Dr. Alberto Zambelli  
ASST Papa Giovanni XXIII 
Piazza OMS, 1 
24127 Bergamo   
 
Prof Salvatore Siena 
Ospedale Niguarda 
ASST Grande Ospedale Metropolitano Niguarda 
Piazza Ospedale Maggiore, 3 
20162 Milano 
 
Prof Francesco Di Meco 
Istituto Neurologico Carlo Besta Milano 
Via Giovanni Celoria, 11 
20133 Milano 
 
 
 
 
With free support of MEDnoTE that provided kits and High Research for its consultant and 
organization of the Performance Study Plan. 
 
 

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PERFORMANCE STUDY PLAN SIGN-OFF PAGE 
 
I have read and understood protocol and I agree to conduct the study as detailed herein and in 
compliance with ICH-GCP and applicable ethical requirements  
 
Prof Giuseppe CURIGLIANO MD PhD 
 
Coordinating Investigator (printed name) 
 
 
 
 
 
 
03 APR 2020 
Coordinating Investigator signature 
 
Date dd-MON-yyyy 
 
 
 
 
Giuseppe CURIGLIANO 
 
Investigator (printed name) 
 
 
 
 
 
 
03 APR 2020 
Investigator signature 
 
Date dd-MON-yyyy 
 
 
 

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Study Type: Observational 
 
Estimated Enrollment: 1000 participants (200 per site) 
 
Observational Model: Cohort 
 
Time Perspective: Prospective 
 
Timelines:  
Actual Study Start Date: April 15, 2020 
Estimated Primary Completion Date: July 31, 2020 
Estimated Study Completion Date: December 31, 2020 
 
 

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Introduction 
The 2019-nCoV IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) is a qualitative membrane based 
immunoassay for the detection of IgG and IgM antibodies to 2019-nCoV in whole blood, serum or plasma 
specimen. This test consists of two components, an IgG component and an IgM component. In the IgG 
component, anti-human IgG is coated in IgG test line region. During testing, the specimen reacts with 2019-
nCoV antigen-coated particles in the test cassette. The mixture then migrates upward on the membrane 
chromatographically by capillary action and reacts with the anti-human IgG in IgG test line region, if the 
specimen contains IgG antibodies to 2019-nCoV. A colored line will appear in IgG test line region as a result 
of this. Similarly, anti-human IgM is coated in IgM test line region and if specimen contains IgM antibodies to 
2019-nCoV, the conjugate-specimen complex reacts with anti-human IgM. A colored line appears in IgM test 
line region as a result. Therefore, if the specimen contains 2019-nCoV IgG antibodies, a colored line will 
appear in IgG test line region. If the specimen contains 2019-nCoV IgM antibodies, a colored line will appear 
in IgM test line region. If the specimen does not contain 2019-nCoV antibodies, no colored line will appear in 
either of the test line regions, indicating a negative result. To serve as a procedural control, a colored line will 
always appear in the control line region, indicating that the proper volume of specimen has been added and 
membrane wicking has occurred. 
Figure 1: Test principle 
 
 
As shown in illustration in Fig. 1, the specimen (A) migrates via capillary action along the membrane to react 
with the gold conjugate (B). 2019-nCoV IgG or/and IgM present in the specimen binds to the conjugate, 
forming a colored Novel coronavirus antibody-antigen complex. The mouse anti-human IgG and mouse anti-
human IgM immobilized in the test zone of the membrane captures the test region (C) and test region (D). 
The formation of a visible colored line in the test region indicates a positive result (C) or (D). The absence of 
a colored line in the test zones suggests a negative result. In the control zone of the membrane, immobilized 
reagents capture colored conjugate regardless of test specimen composition. The resulting visible colored 
band (E) confirms control line. 
Incubation Time: RESULTS AT 10 MINUTES 
Storage: 
Store the test at 2-30°C. Do not freeze. 
 

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Active ingredient list: 
• Mouse anti-human IgG (capture reagent) 
• Mouse anti-human IgM (capture reagent) 
• Mouse IgG 
• Goat anti-mouse IgG 
• 2019-nCov antigen (detection reagent) 
Inactive ingredients list: 
• Adhesive plastic backing 
• Buffer 
• Label pad 
• Absorbent pad 
• Sample pad 
• Nitrocellulose membrane 
• Desiccant (in pouch) 
• Pouch 
• Sample dropper 
1.2/Assay procedure: 
 
 
1) Wash the patient’s hand with soap and warm water or clean with an alcohol swab. Allow to dry. Fig 
1 and Fig 2. 
2) Massage the hand without touching the puncture site by rubbing down the hand towards the 
fingertip of the middle or ring finger 
3) Puncture the finger with a sterile lancet. Wipe away the first sign of blood. Fig 3. 
4) Gently rub the hand from wrist to palm to finger to form a rounded drop of blood over the puncture 
site. Fig 4. 
5) Add the Fingerstick Whole Blood specimen to the test by using a capillary tube: Touch the end of the 
capillary tube to the blood until filled to approximately 20μL. Avoid air bubbles. Fig 5. 

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6) Place the bulb onto the top end of the capillary tube, then squeeze the bulb to dispense the 
wholeblood to the specimen well of the test cassette. Fig 6. 
7) Dispense two drops of diluent to the specimen well of the test cassette. Fig 7.  
8) Read test results after 10 minutes. Fig 8. 
Precautions: 
Bring tests, specimens, buffer, and/or controls to room temperature (15-30°C) before use. 
Bring the pouch to room temperature before opening. Remove the test cassette from the sealed pouch and 
use it within one hour. 
Place the test cassette on a clean and level surface. 
Testing should be performed immediately after specimen collection. Do not leave the specimens at room 
temperature for prolonged periods. Whole blood collected by venipuncture should be stored at 2-8°C if the 
test is to be run within 2 days of collection. Do not freeze whole blood specimens. Whole blood collected by 
fingerstick should be tested immediately. 
Bring specimens to room temperature prior to testing. Frozen specimens must be completely thawed and 
mixed well prior to testing. Specimens should not be frozen and thawed repeatedly. If specimens are to be 
shipped, they should be packed in compliance with local regulations covering the transportation of etiologic 
agents. 
EDTA K2, Heparin sodium, Citrate sodium and Oxalate potassium can be used as anticoagulants. 
Interpretation of the results: 
IgG POSITIVE:* Two colored lines appear. One colored line should always appear in the control line region 
(C) and another line should be in the IgG line region. 
IgM POSITIVE:* Two colored lines appear. One colored line should always appear in the control line region 
(C) and another line should be in the IgM line region. 
IgG and IgM POSITIVE:* Three colored lines appear. One colored line should always appear in the control 
line region (C) and two test lines should be in the IgG line region and IgM line region. 
*NOTE: The intensity of the color in the test line regions may vary depending on the concentration of 2019-
nCoV antibodies present in the specimen. Therefore, any shade of color in the test line region should be 
considered positive. 
NEGATIVE: One colored line appears in the control line region (C). No line appears in the IgG region and IgM 
region. 
INVALID: Control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are 
the most likely reasons for control line failure. Review the procedure and repeat the test with a new test. If 
the problem persists, discontinue using the test kit immediately and contact your local distributor. 
Limitations: 
The 2019-nCoV IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) is for in vitro diagnostic use only. 
This test should be used for detection of IgG and IgM antibody to 2019-nCoV in whole blood, serum or plasma 
specimens. Neither the quantitative value nor the rate of increase in the concentration of IgG or IgM 
antibodies to 2019-nCoV can be determined by this qualitative test. 

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The 2019-nCoV IgG/IgM Rapid Test Cassette (Whole blood/Serum/Plasma) will only indicate the presence of 
IgG and IgM antibodies to 2019-nCoV in the specimen and should not be used as the sole criteria for the 
diagnosis of 2019-nCoV infections. 
As with all diagnostic tests, all results must be considered with other clinical information available to the 
physician. 
If the test result is negative and clinical symptoms persist, additional follow-up testing using other clinical 
methods is suggested. A negative result at any time does not preclude the possibility of 2019- nCoV infection. 
The hematocrit level of the whole blood can affect the test results. Hematocrit level needs to be between 
25% and 65% for accurate results. 
Performance study 
Accuracy, specificity, sensitivity and correlation 
The 2019-nCoV IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) was compared with a leading 
commercial PCR; the results show that 2019-nCoV IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) 
has a high sensitivity and specificity. Results for IgG and IgM are shown in Table 1 and 2 respectively. 
Table 1: Performance of IgG specimen 
 
Specificity = 98.0% (95%CI: 89.4%~99.9%); 
Sensitivity = 100% (95%CI: 86.0%~100%); 
Accuracy = 98.6% (95%CI: 92.3%~99.96%). 
Table 2: Performance of IgM specimen 
 
Specificity = 96.0% (95%CI: 86.3%~99.5%); 
Sensitivity = 85.0% (95%CI: 62.1%~96.8%); 
Accuracy = 92.9% (95%CI: 84.1%~97.6%). 
The 2019-nCov IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) products have been compared 
with a commercial PCR using clinical specimen. The results show that the relative sensitivity of the 2019-nCov 
IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) is 98.6% and the relative specificity is 92.7%. 
Refer to data on investigator brochure for other technical data related to interference and cross-reactivity. 
 

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Rational of the study 
In late December 2019, several local health facilities reported clusters of patients with pneumonia of 
unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan, 
Hubei Province, China. It is now confirmed that the etiology of this outbreak is a novel coronavirus, namely, 
SARS-CoV-2. Of critical importance is rapid and simple diagnostic method to be used in clinical settings to 
timely inform and refine strategies that can prevent, control, and stop the spread of SARS-CoV-2. The 2019-
nCoV IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) is a qualitative membrane based 
immunoassay for the detection of IgG and IgM antibodies to SARS-CoV-2in whole blood, serum or plasma 
specimen. This test consists of two components, an IgG component and an IgM component. In the IgG 
component, anti-human IgG is coated in IgG test line region. During testing, the specimen reacts with 2019-
nCoV antigen-coated particles in the test cassette. The mixture then migrates upward on the membrane 
chromatographically by capillary action and reacts with the anti-human IgG in IgG test line region, if the 
specimen contains IgG antibodies to SARS-CoV-2.  
Aim of the study 
To validate in a real life study a self diagnostic assay for SARS-CoV-2with the advantages of high speed, simple 
operation and low cost, and overcomes the shortcomings of the existing molecular detection methods. 
Clinical specimens from patients who were suspected of being infected with 2019-nCoV will be used to 
evaluate the performance of the assay. In parallel, we will also use molecular assays for the detection of the 
presence of the viral RNA from nasopharyngeal swabs since PCR is currently the gold standard.  
Primary endpoints 
1) To estimate the rate of SARS-CoV-2 positive cancer patients (undergoing chemotherapy, immunotherapy, 
biological therapy, radiotherapy or experimental treatments or major surgery) and health professionals 
in a comprehensive cancer center or in a cancer setting. Patients positive are serially tested with SARS-
CoV-2  lgM / IgG Rapid Test to evaluate the immune response in IgG negative patients and the reliability 
of the test in those patients who develop clinical signs of SARS-CoV-2 during the trial. 
2) To validate the accuracy of self diagnostic assay by confirmation of IgM and/or IgG positive test with the 
current gold standard RT-PCR for positive nucleic acid detection of new coronavirus from nasopharyngeal 
swabs.  
3) To perform on plasma samples multiplex immunoassay protein quantitation related to markers of 
inflammation and immunity (IL-6, TNF, INFy, IL1, IL-2).  2.5 ml of plasma will be needed.    
Secondary endpoint 
A sample of 20 patients in each Centre will be selected by the physician to be monitor using a new cutting-
edge medical device working the telemedicine approach. A telehealth virtual office visit can be performed 
from the patient's home or workplace, decreasing time spent traveling to visit site, time spent in waiting 
room, and cost to patient. Studying telehealth may improve quality of life in patients during the therapy visit, 
especially in this SARS-CoV-2 pandemia. 
Study Population 
Patients or health professionals already tested positive for the viral RNA from nasopharyngeal swabs by RT-
PCR. Patients or health professional who are suspected of being infected with SARS-CoV-2 in the hospital. 
Patients who are considered at high risk for infection and eligible for active therapy and major surgery.  
 

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Criteria 
Inclusion Criteria: 
1. Suspected cases  
Meet the following 2 at the same time: 
a) Epidemiological history: There was a history of contact with confirmed cases before the onset of 
illness; or subjects with at least one symptom in the last week before accrual in the trial. Subjects 
that have been in contact with people positive for SARS-CoV-2 in the previous 14 days. 
 
b) Clinical manifestations are defined as :  
Fever >37.5°; dry  cough, muscle aches and/or fatigue, anosmia, subjects with respiratory distress 
(Respiratory Rate >25/min or O2 Saturation <92%) 
or imaging characteristics of pneumonia; 
or the total number of white blood cells is normal or decreased with the lymphocyte count  decreased 
in the early stage of onset or there is an abnormal C-Reactive protein. Other symptoms that clinical 
investigator will relate to SARS-CoV-2 infection. 
Subject or cancer patients who have been quarantined for suspect symptoms and have access to 
hospital to continue therapy or to receive major surgery 
 
2. Confirmed cases  
Patients or subjects with positive RT-PCR for SARS-CoV-2. On the basis of meeting the criteria for suspected 
cases, sputum, throat swabs, lower respiratory tract secretions, and other specimens were tested by real-
time RT-PCR for positive nucleic acid detection of new coronavirus; or viral gene sequencing was highly 
homologous with known new coronaviruses. Patients positive are serially tested with SARS-CoV-2  lgM / IgG 
Rapid Test to evaluate the immune response in IgG negative patients and the reliability of the test in those 
patients who develop clinical signs of SARS-CoV-2 during the trial. 
 
3. Patients who are considered at high risk for infection and eligible for active therapy and major surgery 
 
Frailty (age and multiple comorbidities) planned to receive a standard systemic anticancer treatment 
comprising chemotherapy and/or immunotherapy and/or radiation therapy or to receive an 
experimental treatment 
 
Major surgery or surgery after neoadjuvant chemotherapy and or chemo/radiotherapy 
 
Exclusion Criteria: 
1. Ascertained influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, human 
metapneumovirus, SARS coronavirus, and other known other viral pneumonia; 
2. Ascertained mycoplasma pneumoniae, chlamydia pneumonia, and bacterial pneumonia; non-infectious 
diseases such as vasculitis, dermatomyositis, and organizing pneumonia. 
 

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Clinical considerations 
All patients with epidemiologic history and/or clinical manifestations suspected for COVID will be proposed, 
according to the clinical judgement, for RT-PCR test for positive nucleic acid detection, irrespective of 
serological evidence.  
All patients tested as IgM positive will have a confirmatory real-time RT-PCR test for positive nucleic acid 
detection of new coronavirus on sputum, throat swabs, lower respiratory tract secretions, and other 
specimens.  
The 2019-nCov IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) will be repeated in patients IgM 
positive for confirming the subsequent IgG positivity. 
The tests will be performed locally in each hospital laboratory.  
The ASST of Cremona laboratory could perform: 
- 
Dr Sophie Testa, UO Laboratorio Analisi – ASST di Cremona: SARS-CoV-2 IgG/IgM Rapid Test Cassette 
(Whole Blood/Serum/Plasma) also for other sites.  1 ml of plasma will be needed;    
- 
Dr Michele Francaviglia- SMeL849 – ASST di Cremona: multiplex immunoassay protein quantitation 
related to markers of inflammation and immunity (IL-6,TNF, INFy, IL1, IL-2, ect).  2.5 ml of plasma will 
be needed.    
.     
The lab responsible person in Milano IEO is: 
Dr.ssa Rita Passerini 
Divisione di Medicina di Laboratorio 
Istituto Europeo di Oncologia 
 
Statistical Plan 
All data recorded will be summarized in tables, graphs and listings.  In general, categorical data will be 
presented using counts and percentages, whilst continuous variables will be presented using the mean, 
standard deviation, median, minimum, maximum and number of patients.   
In tabulations, denominators for calculation of percentages will be taken as the number of non-missing 
responses. 
Primary end-point, the accuracy of tests, will be sensitivity and specificity and they will be calculated for all 
tests and compared with the appropriate statistical test.  
 
References 
1. World Health Organization (WHO). WHO Statement Regarding Cluster of Pneumonia Cases in 
2. Wuhan, China. Beijing: WHO; 9 Jan 2020. [Accessed 26 Jan 2020]. 
3. Weiss SR, Leibowitz JL. Coronavirus pathogenesis. Adv Virus Res 2011;81:85-164. 
4. Su S, Wong G, Shi W, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. 
Trends Microbiol 2016;24:490-502. 
5. Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17:181-
192. 
6. World Health Organization (WHO). Coronavirus.