key: cord-0739217-6epum63l authors: Li, Xinjie; Yin, Yue; Pang, Ling; Xu, Shuyun; Lu, Fengmin; Xu, Dong; Shen, Tao title: Colloidal gold immunochromatographic assay (GICA) is an effective screening method for identifying detectable anti-SARS-CoV-2 neutralizing antibodies date: 2021-06-06 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.05.080 sha: 4b986ff2842a064cd9c0caf751afbe932ddcff90 doc_id: 739217 cord_uid: 6epum63l Introduction A large number of COVID-19 patients are in recovery and millions of people are vaccinated COVID-19 vaccines globally daily. This calls for a rapid screening strategy of SARS-CoV-2 protective antibodies generated in rehabilitators and vaccinated populations. Methods Serum samples collected over a follow-up period of 6 months from 306 COVID-19 cases discharged from Wuhan Tongji Hospital were analyzed. Anti-S Abs were detected by colloidal gold immunochromatographic assay (GICA) and neutralizing antibodies (nAbs) were detected by chemiluminescent microparticle immunoassay (CMIA). Results Most COVID-19 survivors tested positive for anti-S Abs (83.7%) and nAbs (98.0%) 6 months after being discharged from the hospital, and the levels of anti-S Abs in the blood were highly positively correlated with nAbs (r = 0.652, P < 0.0001). The positivity rate of nAbs for patients with anti-S Abs positive was 100%. Conclusions There is a good agreement between anti-S Abs detected by GICA and nAbs detected by CMIA. It indicates that anti-S Abs detected by GICA may be used as a cheaper screening strategy for detectable SARS-CoV-2 nAbs in COVID-19 convalescent individuals. The ongoing global SARS-CoV-2 pandemic has placed a huge burden on the global public health system and the economy at large. Each day, hundreds of thousands of new confirmed cases burst around the world (WHO, 2021) . Meanwhile, a large number of patients are in recovery, and some of which still at the risk of reinfection after rehabilitation as a consequence of the lack of or inability to produce adequate protective antibodies (Hall et al., 2021 , Lumley et al., 2021 . There have been efforts to develop effective vaccines as the primary means to curtail the detrimental effects of the SARS-Cov-2 infection, and so far, millions of people are vaccinated globally on a daily basis (Chung et al., 2021 , Mohammad, 2021 . To inform vaccination and optimize immunization strategies, rapid assessment of the level and duration of protective antibodies in natural post-infected and recovered patients make sense. Plaque reduction neutralization test (PRNT) and microneutralization assay (NT), the gold standards for determining antibody neutralizing activity against SARS-CoV-2, cannot be widely adopted because of their low flux, time-consuming, and fussy operation (CDC, 2021) . Recently, several companies have developed reagents such as chemiluminescent microparticle immunoassay (CMIA) kits based on the principle of competitive inhibition for the detection of SARS-CoV-2 nAbs. The reagents could be widely used because of their high throughput and sensitivity properties (Bonelli et al., 2020 , Taylor et al., 2021 . However, limited by expensive instruments, it is hard to widely serve in undeveloped countries and regions with backward economies. J o u r n a l P r e -p r o o f Therefore, cheap, accurate, simple, and rapid methods for quantifying serum nAbs in recovered patients are valuable to know the duration of antibody response after infection, which can guide the development and refinement of vaccine and public immunization strategies. Herein, we reported a good agreement between anti-S Abs detected by colloidal gold immunochromatographic assay (GICA) and nAbs detected by CMIA. All positive anti-S Abs results identified by GICA were also nAbs positive, indicating that this method can be used as a cheaper screening strategy for SARS-CoV-2 nAbs. In this study, 306 patients recovered from COVID-19 admitted at the Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China were enrolled. The patients were admitted to the hospital between January and February 2020 and had no reinfection during the follow-up period . The diagnosis was confirmed based on positive high-throughput sequencing of nasopharyngeal swab specimens or nucleic acid detection using real-time reverse transcription-polymerase chain reaction (RT-PCR), according to interim guidelines of the World Health Organization (WHO, 2020). All patients were tested multiple times throughout to ensure diagnostic accuracy. All patients were followed up after discharge to ensure no reinfection occurred. Serum samples were collected six months after discharge for assessment of protective antibody levels. The grading of disease on admission was based on the Chinese management guideline for COVID-19 (version 7.0) (NHC, 2020). Mild cases were defined as patients with mild clinical symptoms and pneumonia manifestation undetectable by imaging examination. Moderate cases were defined as patients presenting with clinical symptoms, and pneumonia features detectable by imaging tools. Severe cases were those presenting with any of the following: respiratory distress with RR ≥ 30 times/min; pulse oxygen saturation (SpO2) ≤ 93% at rest; arterial partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ≤ 300 mmHg (1 mmHg = 0.133 kPa). Critical cases were those showing one of the following features: respiratory failure that needs mechanical ventilation, shock, multiple organ failures that call for monitoring of intensive care unit (ICU), or death. Six months after discharged from the hospital, blood samples were collected from patients undergoing rehabilitation by experienced doctors. The blood samples were centrifuged to obtain serum which was stored at 4°C. All antibody tests were performed within 24 hours after sample collection. The colloidal gold immunochromatographic assay (GICA) reagents (Wondfo, Guangzhou, China, batch number: W19501002), which specifically target the spike glycoprotein (S-protein) antibody were used to measure the total antibody of SARS-CoV-2 in serum (IgM and IgG). Briefly, 10μl serum samples and 80μl diluting water (almost 2-3 drops) were added to the hole of the test card, and the test results were observed after15-20 minutes. If the sample contained IgM or/and IgG antibodies of J o u r n a l P r e -p r o o f COVID-19, the antibodies would bind to the recombinant antigen encapsulated in colloidal gold particles to form a composite substance. Anti-S Abs positive produces a red reaction line in the test area (T) and the quality control area (C). The sensitivity, specificity, positive predictive value, and negative predictive value of total antibody were 86.43%, 99.57%, 93.41%, and 100%, respectively. A colloidal gold immunochromatographic card reader (Hurray Star, Beijing, China) was used to identify C and T-lines, calculate antibody concentration, and determine negative and positive rates. Antibody levels were graded into 5 levels (from negative to ++++), where a negative result was defined as the presence of the C-line and the absence of the T-line, + was a detectable of T-line but faint; ++ was defined as a T-line≤50% intensity of the C-line; +++ was defined as the T-line >50% but ≤100% intensity of the C-line and ++++ was defined as the T-line >100% intensity of the Cline. A typical grading diagram of anti-S Abs detected by GICA is shown in Figure 1 . NAbs were assayed using COVID-19 neutralizing antibody detection kits (Hotgen, Beijing, China, batch number: 21010115) based on chemiluminescent microparticle immunoassay (CMIA). The serum was collected and prepared in the same way as before. All operations were carried out in strict accordance with the instructions of the reagent manufacturer. According to the competing strategies, in short, the test was performed by reacting the sample with alkaline phosphatase (ALP)labelled S-RBD antigen. The mixture was incubated to form a neutralizing antibody S-RBD antigen complex. Biotin-labelled receptor protein ACE2 and magnetic microspheres encapsulated with streptavidin were added to promote attachment of the J o u r n a l P r e -p r o o f ACE2-S-RBD antigen complex to the magnetic microspheres by specific binding of biotin and streptavidin. Matched high throughput automatic chemiluminescence immunoassay analyzer was used to analyze nAb levels which were presented as the chemiluminescence signal values divided by the cutoff (absorbance/cutoff, S/CO). Based on the scanned parameter information in the kit, the analyzer automatically calculated the cutoff (CO) after calibration of Calibration 1 and Calibration 2. Each test consumed about 50ul of serum samples and the result was considered a positive test when the S/CO level was lower than 1. To better display the difference of nAb levels between the recovered COVID-19 patients and healthy people, we included the nAb test results of 200 healthy individuals as healthy controls. The healthy controls had never been exposed to SARS-CoV-2 and were negative for both nucleic acid and antibody tests. Statistical analysis was performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were compared with the χ2 test or Fisher's exact test as appropriate, and were expressed as counts or percentages. Correlation analysis was performed using the Spearman analysis. Variables that did not conform to a normal distribution were presented as the interquartile range (IQR). Figures were plotted using Graphpad Prism 8.0. The median age of patients in the study was 62 years (interquartile 53-68 years) J o u r n a l P r e -p r o o f and 45.1% (138/306) of the patients were male. Among them, 40.5% (124/306) were graded as severe cases whereas 2.6% (8/306) were diagnosed as critical cases, and these two categories were equally distributed between sexes (Table 1) . Six months after being discharged from the hospital, 83.7% ( Table 2 ). The level of anti-S Abs in the blood was highly positively correlated with nAbs (r = 0.652, P <0.0001) ( Figure 2 ). As the level of anti-S Abs fell from ++++ to negative, the S/CO value of nAbs in recovered patients increased progressively, indicating a downward trend in nAbs levels. The S/CO values for HCs as healthy controls were all above 1, which denoted a negative nAbs. Variances in the severity of disease, age, and gender were also observed among various antibody subgroups. In terms of disease severity, patients who tested negative for anti-S Abs and nAbs all presented with moderate disease, while in anti-S Abs and nAbs positive group, patients were mainly severe (54.2%), followed by moderate J o u r n a l P r e -p r o o f (42.6%) and critical (3.2%) (Figure 3b ). Compared to anti-S Abs and nAbs positive group, anti-S Abs and nAbs negative patients were predominantly young and middleaged adults (66.7% vs. 16.8%) (Figure 3a) and male (66.7% vs. 46.9%) (data not shown). After SARS-CoV-2 infection or vaccination, the human immune system produces neutralizing antibodies (mainly anti-RBD) that bind to the S1 RBD epitopes, which hinders the ability of the virus to infect human cells (Garcia-Beltran et al., 2021) . Earlier studies indicated that serum levels of protective antibodies drop sharply during the rehabilitation of patients (Gaebler et al., 2021 , Isho et al., 2020 . This demonstrated that the protective power of antibodies may decrease over time. Although it has been suggested that vaccination results in a higher nAb titer compared to titers generated from recovery sera, the actual efficacy and duration of vaccineinduced antibody response are blurred (Jackson et al., 2020) . The WHO recommends that the duration of protection conferred by the COVID-19 vaccine exceeds 6 months. Consequently, clinical and public health surveillance is required to monitor plasma nAb levels in individuals who have recovered from COVID-19 or have been vaccinated, to develop more effective strategies against reinfection and improve the available vaccines. Currently developed methods and commercially available kits for the evaluation of nAb levels can be classified as fluorescence-based assays, lateral flow J o u r n a l P r e -p r o o f immunoassays (LFA), enzyme-linked immunosorbent assays (ELISA), and chemiluminescent immunoassays (CLIA), most of which exhibit the advantage of high throughput and are dependent on professionals and large, costly equipment , Bonelli et al., 2020 , Muruato et al., 2020 , Taylor et al., 2021 . As a point of care testing (POCT) method, the colloidal gold method has been applied in clinical practice for the diagnosis and post-recovery detection of COVID-19. It also features advantages of sensitive, specific, time-saving, consumes a small amount of specimen, and is easy to perform without the requirement for special equipment, comparing with a variety of other serological testing methods (Fu et al., 2020 , Huang et al., 2020 . Previous studies have demonstrated a correlation between IgG titers and nAb titers in the convalescent plasma of COVID-19 recovered patients, suggesting that IgG levels could predict nAb levels to some extent (Klein et al., 2020 . In our study, anti-S Abs detected by GICA and nAbs detected by CMIA presented a favorable correlation. This presumably on account of the S1-specific antibodies, which were detected by GICA, are intimately related to the virus neutralization response. Conversely, N-specific antibodies detected by other serological assays showed a relatively poor correlation with nAb levels (Luchsinger et al., 2020) . Given the close association between anti-S Abs detected by GICA with nAbs, this method could be suitable for primary screening periodic self-evaluation of the protective effects of nAbs in COVID-19 convalescent individual and post-vaccination populations. The latest research has revealed that low levels of nAb titers in convalescents are sufficient to prevent infection, and even lower titers are required to prevent severe symptoms (Khoury et al., 2021) . For patients who tested negative for anti-S Abs, this result does not mean the protection power disappeared, and additional detections should be considered to further improve the efficiency of tests. When judiciously combined, those methods promise reliable monitoring of protective antibody levels, which is useful for developing strategies to minimize the risk of infection and reinfection. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. TS and XL designed the whole study. XL and YY collected and analyzed the data. XL and YY took the lead in drafting and interpreting the manuscript. TS and DX revised the manuscript. All authors were participated in the development and revision of statistical methods. All authors reviewed and approved the manuscript for publication. J o u r n a l P r e -p r o o f Summary of the Detection Kits for SARS Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19 Clinical and Analytical Performance of an Automated Serological Test That Identifies S1/S2-Neutralizing IgG in COVID-19 Patients Semiquantitatively Antibody Testing COVID-19 vaccines: The status and perspectives in delivery points of view The Utility of Specific Antibodies Against SARS-CoV-2 in Laboratory Diagnosis Evolution of antibody immunity to SARS-CoV-2 COVID-19-neutralizing antibodies predict disease severity and survival SARS-CoV-2 infection rates of antibody-positive compared with antibody-negative health-care workers in England: a large, multicentre, prospective cohort study (SIREN) Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients An mRNA Vaccine against SARS-CoV-2 -Preliminary Report Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population Predictive indicators of severe COVID-19 independent of comorbidities and advanced age: a nested case-control study Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers Immune response scenario and vaccine development for SARS-CoV-2 infection A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood National Health Commission of the People's Republic of China A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected: interim guidance COVID-19) Dashboard; 2021 The authors thank Dr. Jerry for polishing the language during the preparation of this manuscript.