The outbreak of new coronavirus disease (COVID-19) has quickly spread all over the world. Real time reverse transcriptase polymerase chain reaction (rRT-PCR) for nucleic acid detection has become the standard method for clinical diagnosis of COVID-19 infection. But these rRT-PCR tests have many inherent limitations, and carry a high false negative rate. It is an urgent to develop a method to accurately identify the vast infected patients and asymptomatic viral carriers from the population. In this article, we present the principle and procedure of developing a colloidal gold immunochromatographic assay (GICA) for rapid detection of COVID-19-specific antibodies. The detection kit can be used to detect immunoglobulin M (IgM) and IgG of COVID-19 in human blood samples within 15 minutes, and to identify different stages of viral infection. Test results can be digitalized using an office scanner and a FiJi software with appropriate confidence interval (CI) setting. Based on analysis from 375 samples, we calculated that overall sensitivity and specificity of the assay were 95.85% and 97.47%, respectively. Compared with rRT-PCR, this assay has many advantages including convenience and rapid detection. The detection kit can be widely used in hospitals, clinics and laboratories for rapid screening of both symptomatic and asymptomatic COVID-19 carriers in large scale.
We have developed a rapid IgM/IgG antibody detection kit for COVID-19. Sensitivity and specificity of this kit have been evaluated. Combined with our data analysis method, this kit can meet the need for rapid COVID-19 detection in any institution with medium configuration.In this manuscript, the detailed working documents for the preparation of the kit are also provided to facilitate any countries and institutions to develop and manufacture the products earlier to meet the emergency needs of the current global pandemic of COVID-19.Because the COVID-19 recombinant antigen was used in this kit, the test result is fairly specific for COVID-19. At the same time, within the titer ranges of positive samples of the COVID-19 IgM/IgG antibodies, the test results of our products did not show a hook effect. The sensitivity and specificity of this kit can reach 95.85% and 97.47%, respectively. However, false negative results still can be found. We speculate the reasons for the false negative results are as follows: (1) low antibody concentration. When the IgM and IgG levels are below the limit (cutoff value) of this kit, the test result will be assessed as negative.(2) The variation of individual immune response of COVID-19.
According to the experience of SARS-CoV treatment, the IgM antibody is the first defence and can be detected in the serum after 1 to 6 days of infection.13 After 8 days, the most abundant antibody IgG can be detected.13 Based on the results of the patient with moderate clinical symptoms, we speculate that by the 6 days of admission, the IgM level had decreased below the cutoff value (Fig. 4). Since the IgM antibody will decrease and disappear after 2 weeks of infection, the single IgM test will lead to more false negative results and is likely not effective enough. Because the false negative results will bring more opportunities to infect other people, we suggest that the multiple and combined IgM/IgG test should be carry out for the patients and suspected cases, and the quantitative analysis should be conduct simultaneously. It means a lot for infection analysis, avoid false negative results and better control of the transmission.
Because COVID-19 infections originate from lungs rather than the upper respiratory tract,1 at the early stage of infection, the rRT-PCR examination with nasopharyngeal swab or sputum may not correctly detect the nucleic acid of COVID-19, and this may be one of the reasons of the higher false negative rate of rRT-PCR. The rapid COVID-19 IgM/IgG test kit owns multiple advantages: it can be performed at the bedside, in any clinic or laboratory, airport or railway station.15 Compared with complicated sample collection of nasopharyngeal swab or sputum for rRT-PCR, it uses serum or plasma as a test sample, and blood samples collection is much easier than nasopharyngeal swab.16 Therefore, it can greatly reduce the risk of cross infection for medical staff. In addition, it is rapid, simple and fast to operate, does not require expensive equipment or certified laboratories or specific training, making the large scale screening more convenient.
Another potential application of this kit is to screen the asymptomatic COVID-19 carriers. It is reported that asymptomatic carriers can transmit COVID-19 virus constantly,18 and this makes the current COVID-19 outbreak control more complicated because of the lack of convenient test kit or method to discriminate the asymptomatic carriers from large crowds.19 This IgG/IgM rapid test kit can also be applied to large scale screening of patients with asymptomatic infections. Of course, this rapid test cannot replace rRT-PCR in confirmation of COVID-19 infection, but it can be used as an alternative supplement for rRT-PCR. Together with other test results, we speculate that combining the IgM/IgG rapid test with rRT-PCR, it can provide more accurate information for the diagnosis of COVID-19 infection.
Currently, due to the rapid outbreak of COVID-19 and personal behaviour regulation, we are unable to carry out the larger range of research activities to cover all genetic mutation-related antigens, and not yet conducted sufficient tests to verify the cross-reaction with antibodies of other virus infections (such as influenza virus). However, our results have verified that the most common interfering substances such as bilirubin and lipid (triglycerides, cholesterol) will not interfere with the test.
Another significance of this study is the detailed and objective method for non-naked eye assessment. Due to personal experience and subjectivity, it will inevitably cause the false judgments of the test results, especially for the results with low antibody concentrations. We use an office scanner to digitalize the test results, and FiJi software to quantify the test bands. Together with the appropriate confidence interval (CI), it effectively avoids the possible mistakes of naked eye judgment. Of course, if the portable device can be developed to assist the real-time data analysis, it ensured that more patients or suspected cases can be benefit from this kit.
According to current knowledge and information, IVD companies in several countries are developing or have developed similar products, including only IgM and IgM/IgG combination tests. For various reasons, we are unable to obtain more details about these techniques and performances, and there are not enough publications for reference. We believe that with the development of epidemics and better products for clinical use, more publications about COVID-19 examination will be available, which will be very helpful for our subsequent comparative research.
In addition, some limitations must be pointed out in this study. Firstly, for time limited, the performance and efficiency of this product was only verified in serum and plasma, the fingertip blood or venous whole blood have not been fully evaluated yet. Secondly, only a moderate number of samples were tested in this study. Because the number of COVID-19 patients has decreased significantly and scattered in China, larger-scale tests cannot be performed in a short time. At last, the interference validation of potential interfering substances, only a few number cases of bilirubin, cholesterol and triglyceride were verified, the other substances, such as rheumatoid factor (RF) and antinuclear antibody (ANA) on the product are on-going.
We have used the colloidal gold immunochromatography technology to develop a kit for rapid COVID-19 IgM/IgG detection. It only takes 10–15 minutes to generate results that are visible to the naked eye, without special requirements for equipment and personnel. With the simple and easy digitalization method, the test results can be semi-quantified and analysed to determine whether the patient has been infected with COVID-19 recently. The results indicated that this kit is sensitive and specific to COVID-19, and showed huge potential for rapid and large scale screening. At the present stage, it cannot replace rRT-PCR for nucleic acid examination and diagnosis, but it can be used as an effective supplementary test in clinical applications.
Reference & Source Information : https://pubs.rsc.org/
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