Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation

Abstract

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Keywords: COVID-19; Panbio; SARS-CoV-2; antigen; clinical methods; diagnostics; epidemiology; false positive; virology.

FIG 1

Summary and principle of the Panbio COVID-19 Ag rapid test device. (A) Panbio kit components and summary of the test procedure. The Panbio kit is designed for detection of SARS-CoV-2 antigen (i.e., nucleocapsid protein or N protein). Following specimen collection, the swab is placed into an extraction tube prefilled with 11 to 12 drops (or 300 μl) of buffer, and the tube cap is added. The tube is pinched to help extract the respiratory secretions from the swab, which in turn is rotated into the buffer. The nozzle cap is removed from the extraction tube, and 5 drops are placed into the sample well of the Panbio lateral flow device. After 15 to 20 min, the results are read and interpreted as depicted. (B) The principle of the Panbio Ag-RDT relies on a nitrocellulose membrane precoated with anti-chicken IgY at the control line and an anti-SARS-CoV-2-specific antibody at the test line. When the buffer/specimen solution is added to the sample well, the liquid flows progressively through the device using capillary action and sequentially flows through the sample pad, the conjugate pad, the nitrocellulose membrane, and eventually into the wick. As the liquid comes into contact with the conjugate pad, both conjugated antibodies are resuspended (i.e., the gold-conjugated chicken IgY and the gold-conjugated human IgG specific to SARS-CoV-2). In the absence of SARS-CoV-2 antigen (i.e., N protein), the conjugated anti-SARS-CoV-2 antibody will not interact with the anti-SARS-CoV-2 capture antibody at the test line; however, the conjugated chicken IgY will be captured by the anti-chicken IgY immobilized at the control line. This generates a red-colored band that can be visualized. In the presence of SARS-CoV-2 antigen, a similar reaction occurs at the test line due to the interaction between the antigen and the conjugated and capture anti-SARS-CoV-2 antibodies. As seen in this study, false positives can occur with testing conditions falling outside the manufacturer instructions, depicted here as nonspecific interactions between the conjugated and capture anti-SARS-CoV-2 antibodies in the absence of antigen.

FIG 2

False-positive SARS-CoV-2 Ag-RDT results can occur from Panbio buffer absence, dilution, or alterations. (A) Artifact generation by Panbio buffer dilution in PCR-grade water at different temperatures (4°C, 25°C, and 37°C). False-positive SARS-CoV-2 Ag-RDT result occurrence from uncontrolled pH and buffering conditions (B) or from changes in ionic strength from NaCl (C) are shown. Of note, results presented in Fig. 2C correspond to those obtained from solutions of 100 mM tricine, in which different concentrations of NaCl were prepared. All experiments were performed in the absence of SARS-CoV-2 antigen. False-positive reactions are indicated as POS (in red) under each Ag-RDT result; NEG, negative; INV, invalid.

FIG 3

Impact of proteinase K (PK) and heat treatment on the conjugated SARS-CoV-2 antibody. (A) Conjugate pad transplantation was used to access and investigate properties of the proprietary Panbio conjugated antibodies. Each step was followed as depicted, leading to treatment of the conjugated antibodies with proteinase K (PK) or heat (T°C), and comparisons were made with untreated controls (none). In some experiments (dashed arrows), the gold-conjugated antibody suspensions were pretreated with mouse anti-chicken IgY (obtained from a fragment of the nitrocellulose membrane at the control line) to purify the gold-conjugated human IgG specific for SARS-CoV-2 conjugated antibody. This suspension was used for subsequent lateral flow and thermal shift assays; RT, room temperature. (B) PK and heat treatments of the conjugated antibodies. Using conjugate pad transplantation, gold-conjugated antibody suspensions in Panbio buffer or water were treated for 1 h with PK at 56°C, followed by heat inactivation of PK at 70°C for 10 min. Following reintroduction into Panbio cassettes of conjugated antibodies that were untreated (none), heat treated (T°C), or PK treated, water was inoculated in the sample well. (C) Removal of the conjugated chicken IgY from the conjugated antibody suspensions to purify the conjugated SARS-CoV-2-specific antibody. Untreated (none) or pretreatment (−IgY) are depicted for reassembled Panbio cassettes containing the purified conjugated SARS-CoV-2-specific antibody, which was then inoculated with PCR-grade water. For B and C, similar reactions as performed for water were undertaken with a positive- or negative-control swab to demonstrate that the method did not impact conjugate antibody function.

FIG 4

Thermal shift profiles for the Panbio gold-conjugated human IgG specific to SARS-CoV-2 at different pH values. All reactions were performed in 100 mM tricine, and representative thermal shift profiles are presented (A). Melting temperature (T_m) changes based on pH are summarized (B) as well as which conditions were consistent (i.e., pH 5 to 7) or inconsistent (i.e., pH 8 to 10) with the generation of false-positive results when tested by Ag-RDT.