Distribution characteristics of SARS-CoV-2 IgM and IgG in false-positive results detected by Chemiluminescent immunoassay

There have been several false-positive results in the antibody detection of the COVID-19. This study aims to analyze the distribution characteristics of SARS-CoV-2 IgM and IgG in false-positive results detected using chemiluminescent immunoassay. The characteristics of the false-positive results in SARS-CoV-2 IgM and IgG testing were retrospectively analyzed. The dynamic changes in the results of SARS-CoV-2 IgM and IgG antibodies were observed. The false-positive proportion of the single SARS-CoV-2 IgM positive results was 95.88%, which was signi�cantly higher than those of the single SARS-CoV-2 IgG positive results (67.50%) (P < 0.001) and SARS-CoV-2 IgM & IgG positive results (29.55%) (P < 0.001). The S/CO of the SARS-CoV-2 IgM and IgG in false-positive results ranged from 1.0 to 50.0. The false-positive probability of SARS-CoV-2 IgM in the S/CO range (1.0 ~ 3.0) was 91.73% (77/84), and the probability of false-positive of SARS-CoV-2 IgG in the S/CO range (1.0 ~ 2.0) was 85.71% (24/28). Dynamic monitoring showed that the S/CO values of IgM in false-positive results decreased or remained unchanged, whereas the S/CO values of IgG in false-positive results only decreased. The possibility of false-positive of the single SARS-CoV-2 IgM positive and single SARS-CoV-2 IgG positive results was high. As the value of S/CO decreased, the probability of false-positive consequently increased, especially among the single SARS-CoV-2 IgM positive results.


Introduction
The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a catastrophic effect on the world's demographics, becoming the most consequential global health crisis since the in uenza pandemic of 1918 [1][2][3][4] .Since the onset of the pandemic, rapid and accurate diagnoses have helped in epidemiologic monitoring, effective preventive measures, and appropriate antiviral therapies.Nucleic acid tests (N.A.T.s) have been widely used to detect viral infections as con rmed etiological evidence for suspected COVID-19 patients 5 .However, a signi cant limitation in N.A.T.s is the high false-negative rate, owing to various factors, including improper sampling, sample types, viral mutation, and patient viral load [6][7][8][9][10][11] .Moreover, N.A.T.s require high-quality environmental conditions and equipment, and its detection procedure is time-consuming and labor-intensive, in addition to its already limited sensitivity [8][9][10][11] .
Aside from nucleic acid detection, assay kits for detecting IgM and IgG antibodies against SARS-CoV-2 proteins have expanded our measures for COVID-19 detection.Compared with N.A.T.s, serological tests have a faster turnaround time, signi cantly reducing the risk of infection among medical staff during pharyngeal swab sampling.Some studies have reported that almost all COVID-19 patients develop detectable IgG and IgM antibodies within several weeks of symptom onset [12][13][14][15] .Studies have also shown that serology-based diagnosis methods can be used with N.A.T.s to improve detection accuracy and exclude false-negative results [14][15][16][17][18] .Furthermore, the detection of speci c antibodies has been proven to show public health and clinical utility for pandemic monitoring and response and managing affected patients 18 .
However, it should be noted that overdiagnosis in false-positive results can lead to an incorrect conclusion that an individual has been infected, thereby causing psychological damage and unnecessary nancial losses for community isolation and contact tracing 23 .Given antibodies detection's role in the pandemic response, clinicians and technicians must practice vigilance in possible false-positive results.Furthermore, our previously published work 24 showed that false-positive SARS-CoV-2 IgM results could be caused by a moderate to a high concentration of rheumatoid factor IgM (RF-IgM) in the patient's serum.Thus, from a practical perspective and to combat the increasing number of COVID-19 cases, a timely and effective way to check the authenticity of screening positive results is needed.
We retrospectively analyzed the cases with false-positive results in SARS-CoV-2 IgM and IgG testing using the C.I.A. method.We analyzed the characteristics of false-positive results to distinguish false-positive results from true-positive ones.Moreover, a reference for strategizing and guidance may be provided for other researchers.

Materials And Methods
Subjects and case de nition.This was a retrospective study involving the data of patients who tested for SARS-CoV-2 infection in the outpatient and inpatient departments of the A liated Hospital of North Sichuan Medical College and Nanchong Central Hospital, Nanchong, China.
For this study, clinical classi cation, clinical manifestations, personal demographics, and laboratory ndings were obtained from the electronic medical records.When the initial test of the SARS-CoV-2 speci c antibody was positive, patient records regarding SARS-CoV-2 infection were retrospectively reviewed in duplicates by two or more physicians independently to determine whether each case did have a SARS-CoV-2 infection.A con rmed COVID-19 case was de ned based on the Diagnosis and Treatment Protocol for COVID-19 (7th edition), released by the National Health Commission of the People's Republic of China 5 .When the diagnostic criteria were ful lled, the case was true SARS-CoV-2 infection, including a case of current SARS-CoV-2 infection or resolved SARS-CoV-2 infection.On the other hand, a case that was clinically excluded for COVID-19 or was vaccinated against COVID-19 was no evidence of SARS-CoV-2 infection.Moreover, a false-positive case was de ned as a positive antibody result with no evidence of SARS-CoV-2 infection.
Measurement of SARS-CoV-2 IgM and IgG.Serum levels of the SARS-CoV-2 IgM and IgG were determined using the automatic C.I.A. system (Bioscience Biotechnology Co., Ltd) with reagents, including the SARS-CoV-2 antibodies (IgM and IgG) detection kits (Bioscience Biotechnology Co., Ltd, lot number of IgM: G202002415, IgG: G202002414).Brie y, IgG and IgM antibody detection was developed based on magnetic particle chemiluminescence immunoassay (MCLIA), which uses recombinant antigens containing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, acting as the conjugated antigen.The tests were conducted on an automated magnetic chemiluminescence analyzer, and antibody levels were expressed by the chemiluminescence signal.The commercially available assay used in this study has been evaluated to be su ciently sensitive and speci c for detecting SARS-CoV-2 IgM and IgG in clinical specimens 25 .According to the manufacturer's instructions and standard operating procedures, the daily maintenance was operated before sample testing, and both internal quality control and sample testing were carried out afterward.
Samples showing initial positive antibody test results were retested when there was no apparent history of SARS-CoV-2 infection or when the case had no previous laboratory test results, including SARS-CoV-2 IgM and IgG detection and SARS-CoV-2 R.N.A. testing.
Result judgment.SARS-CoV-2 IgM and IgG results detected via chemiluminescent immunoassay were given in the form of the ratios of specimen signals to the cut-off values (S/CO), which were considered to be negative if S/CO < 1.0 and positive if S/CO ≥ 1.0.Statistical Analysis.Data were classi ed and counted using the Excel 2007 software.Measurement data were expressed as means ± S.E.M.s, whereas count data were expressed as percentages.The Chi-square test was used to compare the enumeration data.All statistical analyses were performed using the SPSS version 23.0 program (SPSS Co., Inc., Chicago, IL), and statistical signi cance was de ned at p < 0.05, as determined by two-tailed tests.
Ethical approval and informed consent.The study protocol was approved by the Ethics Committee of the A liated Hospital of North Sichuan Medical College.All experimental protocols and methods were carried out by the relevant guidelines and regulations, and they complied with the principles of the Declaration of Helsinki.Since the samples included in the study were conducted anonymously, the ethics committee of the A liated Hospital of North Sichuan Medical College waived the need for written informed consent.

Results
A total of 14,708 patients were tested with IgM and IgG against SARS-CoV-2 from March 2020 to January 2021, excluding cases with duplicated patients.181(1.23%)patients with positive results (S/CO ≥ 1.0) were included in this study.Among these 181 cases, 48(26.52%)were de ned as "true positive, " the remaining 133(73.48%)were de ned as " false positive  no signi cant difference was found in the false-positive proportion of the three result patterns between different sexes (p > 0.05) (Table 2).Similarly, no signi cant difference was also found in the false-positive rates of the three result patterns in different age groups (p > 0.05) (Table 3).The S/CO values of the SARS-CoV-2 IgM true-positive cases ranged from 1.0 to 180.0, with most of them between 3.0 and 10.0 (Fig. 1A), whereas the S/CO values of single SARS-CoV-2 IgM true-positive cases all were more than 100.0 (Fig. 1B).The overlapping range of S/CO values for SARS-CoV-2 IgM false-positive and true-positive cases was mainly from 1.0 to 50.0 (Fig. 1A).Notably, neither SARS-CoV-2 IgM nor single SARS-CoV-2 IgM in false-positive cases showed S/CO values greater than 40.0 (Fig. 1A, Fig. 1B).
As shown in Table 4, when the S/CO values of the SARS-CoV-2 IgM were in the ranges of 1.0 ~ 3.0, 3.0 ~ 5.0, 5.0 ~ 10.0, 10.0 ~ 50.0, and greater than 50.0, the probability of SARS-CoV-2 IgM false-positive was 91.73%, 52.63%, 38.89%, 75.00%, and 0.00%, respectively.Meanwhile, when the S/CO values of the single SARS-CoV-2 IgM were in the ranges of 1.0 ~ 50.0 and greater than 50.0, the probability of the single SARS-CoV-2 IgM false-positive was 100.00% and 0.00%, respectively.5).> 50.0 0.00% (0/10) -Eighteen SARS-CoV-2 IgM false-positive cases and four true-positive cases were dynamically monitored at different time points.Of the 18 false-positive cases, 10 cases turned negative, and the remaining eight cases did not.The median time of conversion to seronegative of the 10 cases was nine days.Compared with the dynamic change trend of true-positive cases, the dynamic change trend in SARS-CoV-2 IgM falsepositive cases was signi cantly lower.On the other hand, among the eight false-positive cases that were not monitored to seronegative, three cases showed a decreasing trend, whereas ve remained unchanged (Fig. 3A).
Seven SARS-CoV-2 IgG false-positive cases and four true-positive cases were dynamically monitored at different time points.Of the seven false-positive cases, six turned seronegative, while only one case did not.The median time of conversion to seronegative of the six cases was ve days.Compared with the dynamic change trend of SARS-CoV-2 IgG in true-positive cases, the dynamic change trend in falsepositive cases was signi cantly lower.Notably, the only one false-positive case which was not monitored to seronegative showed a decreasing trend (Fig. 3B).

Discussion
Generally, coronavirus genomes encode four major structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N).Current serologic tests have been developed to target antibodies directed against these antigens, showing that spike protein-based detection was more sensitive than nucleocapsid protein-based detection 26,27 .In our study, we used the C.I.A. method, which was based on the recombinant SARS-CoV-2 S-RBD protein to detect serum IgG and IgM, and its analytical performance was successfully evaluated by Wan Y et al.They reported the performance veri cation of the SARS-CoV-2 IgM (82% sensitivity and 93.85% speci city) and SARS-CoV-2 IgG (86% sensitivity and 96.92% speci city) detection kits among COVID-19 patients 25 .
However, due to the problems of immunological detection methods, there have been interferences attributed to certain pathological factors, biological factors, and cross-reactions, resulting in false-positive results that were inconsistent with clinical manifestations and epidemiological characteristics.Some of these factors identi ed by previous studies included inadequacy during any step of the testing process, presence of cross-reactive antibodies, other endogenous interference factors, and other viral infections 24,28−29 .Additionally, some false-positive cases likely did not result from problems with the sample, procedure, or other random factors, which was supported by obtaining repeated positive results with similar S/CO values on repeat testing (data was not shown) in our study, making a transient response to antigen less likely.Furthermore, this phenomenon regarding endogenous interference factors in SARS-CoV-2 antibody testing was also reported in our previous study 24 .Although we can use the electronic medical records and laboratory results, including N.A.T.s, as a source to determine true anti-SARS-CoV-2 status, the procedure is labor-intensive and time-consuming.Therefore, we attempted to seek an effective strategy to solve such problems when confronted with false-positive results in the SARS-CoV-2 antibody screening test.
To elucidate these issues, we retrospectively analyzed the false-positive cases of SARS-CoV-2 IgM, and IgG detected using C.I.A.This study showed that the false-positive rate of the single SARS-CoV-2 IgM positive results was 95.88%, which was signi cantly higher than those of the single SARS-CoV- Despite the ndings of our study, several limitations were noted.First, due to the insu cient conditions of our laboratory, we failed to determine the interferences or factors causing false-positive results.Second, the number of samples included in this study was limited, leading to some deviation in the analysis results.More cases should be included in further studies of the same topic.Third, since the majority of the SARS-COV-2 speci c antibody detection was performed using the C.I.A. platform due to its high throughput, our research analysis was only focused on the C.I.A. Methods and molecules used for generating and detecting signals.The epitopes and speci cities of antigens and antibodies are different between the assays.Thus, the characteristics of the false-positive results analyzed in this study may not apply to other SARS-COV-2 antibody detection methods.Regardless, this study can still provide a reference strategy for other researchers.

Conclusion
We proposed that the possible usage of the SARS-COV-2IgM and IgG antibody patterns, S/CO values or ranges, and dynamic changes in antibody levels is of great signi cance in screening positive antibody results.This study aimed to assist clinicians or technicians in developing timely and effective diagnostic strategies to distinguish false-positive results from true-positive ones.Thus, if the number of false positives can be reduced with these suggested adjustments, this would have implications for other COVID-19 screening tests.

Declarations
Figures

Figure 1
shows the distribution of SARS-CoV-2 IgM S/CO values in the false-positive and true-positive results.The S/CO values of IgM false positives ranged from 1.0 to 32.0, with most of them concentrated between 1.0 and 3.0 (Fig.1A, Fig.1B).

2
IgG positive results (67.50%) and SARS-CoV-2 IgM & IgG positive results (29.55%).Therefore we concluded that the possibility of false-positive of the single SARS-CoV-2 IgM positive and single SARS-CoV-2 IgG positive results was high, and the combined detection of SARS-CoV-2 IgM and IgG antibody was better than the single detection in terms of a positive detection.Previous investigations have shown that SARS-CoV-2 IgM and IgG antibodies could be detected as early as the 4th day following symptom onset30 .The positive rates of the single SARS-CoV-2 IgM, single SARS-CoV-2 IgG, and SARS-CoV-2 IgM and IgG positive results among COVID-19 patients were 1.72%, 3.45%, and 94.83%, respectively, concluding that the combined detection of IgM and IgG had better practicability and sensitivity than IgM or IgG alone31 .Interestingly, most false-positive signals were detected in the SARS-CoV-2 IgM assays, which other studies have also noted28,32 .Thus, the combined detection of SARS-CoV-2 IgM and IgG should be given high priority in its implementation as the standard serological test in clinical and public health practice during the pandemic.In this study, we found that the S/CO values of the IgM false-positive results were mainly between 1.0 and 3.0, whereas the S/CO values of the IgG false-positive results were mainly between 1.0 and 2.0.These results indicated that the SARS-CoV-2 IgM and IgG false-positive results detected by C.I.A. mainly existed in the low-value area.The false-positive results, which were low positive or low-value, needed to be con rmed further.Therefore, the S/CO ratio may be a helpful indicator in differentiating false positives from true positives.Aside from the S/CO values, we also compared the false-positive proportions of the single SARS-CoV-2 IgM, single SARS-CoV-2 IgG, and SARS-CoV-2 IgM & IgG positive results in different sexes and ages.Our study showed no signi cant differences for the false-positive proportion in different sexes and ages.status shows a rapidly decreasing trend in dynamic monitoring, which may indicate a false-positive result.

Figure 1 Distribution
Figure 1

Figure 2 Distribution
Figure 2

Table 2
Comparison of the false-positive proportion of the three patterns in different sexesAmong the cases in our study, 141 had the S/CO values of SARS-CoV-2 IgM greater than or equal to 1.0.97/141(68.79%)cases had the single SARS-CoV-2 IgM positive results.

Table 4
The probability of the false-positive results in SARS-CoV-2 IgM and single SARS-CoV-2 IgM S/CO = ratios of specimen signals to the cut-off values

Table 5
The probability of the false-positive results in SARS-CoV-2 IgG and single SARS-CoV-2 IgG S/CO = ratios of specimen signals to the cut-off values