Immunoglobulin M Capture Enzyme-linked Immunosorbent Assay Research Articles

Evaluation of Serologic Cross-Reactivity Between Dengue Virus and SARS-CoV-2 in Patients with Acute Febrile Illness - United States and Puerto Rico, April 2020-March 2021.

The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC).† Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.

Detection and confirmation of dengue pre- and postintroduction of dengue NS1-antigen test at the University Malaya Medical Centre: An observational study.

Early diagnosis of dengue is important to ensure proper management of patients and effective implementation of control measures. The present study was undertaken to determine the outcome of the implementation of dengue NS1-antigen (Ag) rapid diagnostic test (RDT) in the confirmation of dengue at the first patient hospital visit at the University Malaya Medical Centre. A total of 1036 and 1097 sera from the year 2008 and 2015 were used, representing samples from before and after dengue NS1-Ag RDT was implemented as routine diagnostic at the hospital. Results showed that similar dengue confirmation percentage (56%) was made in 2008 and 2015, regardless of the main laboratory diagnostic method used. Confirmation of dengue, however, increased to 68% and 73% when dengue NS1-Ag test or dengue immunoglobulin M-capture enzyme-linked immunosorbent assay was used as the second test for the 2008 and 2015 samples, respectively. Detection of dengue virus (DENV) using multiplex reverse transcription-polymerase chain reaction (RT-PCR) showed that DENV-1 was the highest in circulation in 2008 and that both DENV-1 and DENV-2 were dominant in 2015. In summary, the present study demonstrated that the introduction and use of the dengue NS1-Ag RDT did not change or compromise confirmation of dengue, highlighting the advantage of using the method. With the reducing cost of molecular detection tools, DENV detection using RT-PCR remains a viable option for further confirmation of dengue in hospital settings.

Detection of chikungunya virus‐specific IgM on laser‐cut paper‐based device using pseudo‐particles as capture antigen

The incidence of arbovirus infections has increased dramatically in recent decades, affecting hundreds of millions of people each year. The Togaviridae family includes the chikungunya virus (CHIKV), which is typically transmitted by Aedes mosquitoes and causes a wide range of symptoms from flu-like fever to severe arthralgia. Although conventional diagnostic tests can provide early diagnosis of CHIKV infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop efficient, affordable, simple, rapid, and robust diagnostic tools that can be used in point-of-care settings. Early diagnosis is crucial to improve patient management and to reduce the risk of complications. A glass-fiber laser-cut microfluidic device (paper-based analytical device [PAD]) was designed and evaluated in a proof of principle context, for the analysis of 30 µL of patient serum. Biological raw materials used for the functionalization of the PAD were first screened by MAC-ELISA (IgM capture enzyme-linked immunosorbent assay) for CHIKV Immunoglobulin M (IgM) capture and then evaluated on the PAD using various human samples. Compared with viral lysate traditionally used for chikungunya (CHIK) serology, CHIKV pseudo-particles (PPs) have proven to be powerful antigens for specific IgM capture. The PAD was able to detect CHIKV IgM in human sera in less than 10 minutes. Results obtained in patient sera showed a sensitivity of 70.6% and a specificity of around 98%. The PAD showed few cross-reactions with other tropical viral diseases. The PAD could help health workers in the early diagnosis of tropical diseases such as CHIK, which require specific management protocols in at-risk populations.

Random Forest-Based Evaluation of Raman Spectroscopy for Dengue Fever Analysis.

This work presents the evaluation of Raman spectroscopy using random forest (RF) for the analysis of dengue fever in the infected human sera. A total of 100 dengue suspected blood samples, collected from Holy Family Hospital, Rawalpindi, Pakistan, have been used in this study. Out of these samples, 45 were dengue-positive based on immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) tests. For highlighting the spectral differences between normal and infected samples, an effective machine learning system is developed that automatically learns the pattern of the shift in spectrum for the dengue compared to normal cases and thus is able to predict the unknown class based on the known example. In this connection, dimensionality reduction has been performed with the principal component analysis (PCA), while RF is used for automatic classification of dengue samples. For the determination of diagnostic capabilities of Raman spectroscopy based on RF, sensitivity, specificity, and accuracy have been calculated in comparison to normally performed IgM capture ELISA. According to the experiment, accuracy of 91%, sensitivity of 91%, and specificity of 91% were achieved for the proposed RF-based model.

Serological Response to Treatment of Syphilis According to Disease Stage and HIV Status

Serology is the mainstay for syphilis diagnosis and treatment monitoring. We investigated serological response to treatment of syphilis according to disease stage and HIV status. A retrospective cohort study of 264 patients with syphilis was conducted, including 90 primary, 133 secondary, 33 latent, and 8 tertiary syphilis cases. Response to treatment as measured by the Venereal Disease Research Laboratory (VDRL) test and a specific IgM (immunoglobulin M) capture enzyme-linked immunosorbent assay (ELISA; Pathozyme-IgM) was assessed by Cox regression analysis. Forty-two percent of primary syphilis patients had a negative VDRL test at their diagnosis. Three months after treatment, 85%-100% of primary syphilis patients had reached the VDRL endpoint, compared with 76%-89% of patients with secondary syphilis and 44%-79% with latent syphilis. In the overall multivariate Cox regression analysis, serological response to treatment was not influenced by human immunodeficiency virus (HIV) infection and reinfection. However, within primary syphilis, HIV patients with a CD4 count of <500 cells/μL had a slower treatment response (P = .012). Compared with primary syphilis, secondary and latent syphilis showed a slower serological response of VDRL (P = .092 and P < .001) and Pathozyme-IgM tests (P < .001 and P = .012). The VDRL should not be recommended as a screening test owing to lack of sensitivity. The syphilis disease stage significantly influences treatment response whereas HIV coinfection only within primary syphilis has an impact. VDRL test titers should decline at least 4-fold within 3-6 months after therapy for primary or secondary syphilis, and within 12-24 months for latent syphilis. IgM ELISA might be a supplement for diagnosis and treatment monitoring.

Identification of Potential Serodiagnostic and Subunit Vaccine Antigens by Antibody Profiling of Toxoplasmosis Cases in Turkey

Toxoplasmosis, caused by infection of the protozoan parasite Toxoplasma gondii, is associated with mild disease in healthy individuals, whereas individuals with depressed immunity may develop encephalitis, neurologic disorders, and other organ diseases. Women who develop acute toxoplasmosis during pregnancy are at risk of transmitting the infection to the fetus, which may lead to fetal damage. A diagnosis is usually confirmed by measuring IgG, or IgM where it is important to determine the onset of infection. A negative IgM result essentially excludes acute infection, whereas a positive IgM test is largely uninterpretable because IgM can persist for up to 18 months after infection. To identify antigens for improved diagnosis of acute infection, we probed protein microarrays displaying the polypeptide products of 1357 Toxoplasma exons with well-characterized sera from Turkey. The sera were classified according to conventional assays into (1) seronegative individuals with no history of T. gondii infection; (2) acute infections defined by clinical symptoms, high IgM titers, and low avidity IgG; (3) chronic/convalescent cases with high avidity IgG but persisting IgM; (iv) true chronic infections, defined by high avidity IgG and no IgM. We have identified 38 IgG target antigens and 108 IgM target antigens that can discriminate infected patients from healthy controls, one or more of which could form the basis of a 'tier-1' test to determine current or previous exposure. Of these, three IgG antigens and five IgM antigens have the potential to discriminate chronic/IgM persisting or true chronics from recent acutely infected patients (a 'tier-2' test). Our analysis of the antigens revealed several enriched features relative to the whole proteome, which include transmembrane domains, signal peptides, or predicted localization at the outer membrane. This is the first protein microarray survey of the antibody response to T. gondii, and will help in the development of improved serodiagnostics and vaccines.

Early Diagnosis of Dengue Infection Using a Commercial Dengue Duo Rapid Test Kit for the Detection of NS1, IGM, and IGG

A commercial Dengue Duo rapid test kit was evaluated for early dengue diagnosis by detection of dengue virus NS1 antigen and immunoglobulin M (IgM)/IgG antibodies. A total of 420 patient serum samples were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR), in-house IgM capture enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition assay, and the SD Dengue Duo rapid test. Of the 320 dengue acute and convalescent sera, dengue infection was detected by either serology or RT-PCR in 300 samples (93.75%), as compared with 289 samples (90.31%) in the combined SD Duo NS1/IgM. The NS1 detection rate is inversely proportional, whereas the IgM detection rate is directly proportional to the presence of IgG antibodies. The sensitivity and specificity in diagnosing acute dengue infection in the SD Duo NS1/IgM were 88.65% and 98.75%, respectively. The assay is sensitive and highly specific. Detection of both NS1 and IgM by SD Duo gave comparable detection rate by either serology or RT-PCR.

Naturally Acquired IgM Antibody Response to the C-Terminal Region of the Merozoite Surface Protein 1 of Plasmodium vivax in Korea: Use for Serodiagnosis of Vivax Malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-naïve healthy individuals (control group 1), and 70 malaria-naïve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.

Immunoglobulin M‐Capture Enzyme‐Linked Immunosorbent Assay Testing of Cerebrospinal Fluid and Serum from Horses Exposed to West Nile Virus by Vaccination or Natural Infection

The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti‐WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood‐brain barrier (BBB) in WN virus‐infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto‐occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM‐capture‐enzyme‐linked immunosorbent assay (MAC‐ELISA) was used to detect anti‐WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P &lt; .02) IgG index compared to the AO site (0.34±0.04 versus 0.22±0.04 [mean ± SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC‐ELISA testing. However, the positive to negative antigen ratios were higher (P &lt; .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC‐ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.

Utility of the focus technologies west nile virus immunoglobulin M capture enzyme-linked immunosorbent assay for testing cerebrospinal fluid.

Focus Technologies has developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) kit that utilizes recombinant West Nile virus (WNV) antigens to detect WNV IgM in serum. We evaluate here the utility of the kit for detecting WNV IgM in cerebrospinal fluid (CSF). The sensitivity was evaluated by using 52 CSF specimens from the 2002 WNV season that were positive in both the Public Health Service Laboratories WNV IgM ELISA and an in-house WNV IgM ELISA with native WNV antigen. The specificity was evaluated with two groups of specimens: (i). 73 CSF specimens submitted for in-house WNV IgM ELISA testing from February through April 2003 and yielding a negative WNV IgM result and (ii). 60 CSF specimens determined to be positive for another virus by PCR testing. Using these 185 CSF specimens at a screening dilution of 1:2, the kit was determined to be 100% sensitive and 100% specific. Endpoint titers were determined for 20 IgM-positive CSF specimens by testing serial twofold dilutions and ranged from 1:8 to 1:512. Index values (specimen absorbance value/calibrator absorbance value) for the screening dilution (1:2) showed no correlation with IgM titers, whereas index values for higher dilutions showed significant correlation with IgM titers. CSF screening dilutions of greater than 1:2 are not recommended, however, due to the risk of obtaining false-negative results. These findings show that the Focus Technologies WNV IgM capture ELISA, when utilized as recommended, offers accurate qualitative detection of WNV IgM in CSF specimens.

Immunoglobulin M antibody test to detect genogroup II Norwalk-like virus infection.

Sera obtained from adult volunteers inoculated with genogroup II Norwalk-like viruses (NLVs), Hawaii virus, and Snow Mountain virus and from patients involved in outbreaks of gastroenteritis were tested for genogroup II NLV Mexico virus-specific immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant Mexico virus antigen (rMXV)-based IgM capture enzyme-linked immunosorbent assay (ELISA). Sera from genogroup I Norwalk virus (NV)-inoculated volunteers and from patients involved in a genogroup I NLV outbreak were also tested. In sera from those infected with genogroup I NV or NLVs in volunteer and outbreak studies, only 3 of 25 were rMXV IgM positive; in contrast, 24 of 25 were IgM positive for recombinant NV (rNV). In sera from those infected with genogroup II NLVs in volunteer and outbreak studies, 28 of 47 were rMXV IgM positive and none were IgM positive for rNV, showing the specificity of each IgM test for its respective genogroup. In an outbreak of gastroenteritis not characterized as being of viral etiology but suspected to be due to NV, 7 of 13 persons had IgM responses to rMXV, whereas none had IgM responses to rNV, thus establishing the diagnosis as genogroup II NLV infection. The rMXV-based IgM capture ELISA developed is specific for the diagnosis of genogroup II NLV infections.

Detection of specific antibodies in saliva during dengue infection.

Saliva was collected prospectively from patients presenting with suspected dengue infection 4 to 8 days after the onset of symptoms and assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA). Laboratory diagnosis was based on virus isolation and on hemagglutination inhibition (HAI) assay and an in-house IgM and IgG capture ELISA. With a positive result defined as either salivary IgM or IgG levels above the cutoff value, an overall sensitivity of 92% was obtained for both primary- and secondary-dengue patients (22 of 24), while no patients with non-flavivirus infections (n = 11) and no healthy laboratory donors (n = 17) showed elevation of salivary antidengue antibody (100% specificity). Salivary IgG levels correlated well with serum HAI titer (r = 0.78), and salivary IgG levels could be used to distinguish between primary- and secondary-dengue virus infections.

Rapid diagnosis of Japanese encephalitis by using an immunoglobulin M dot enzyme immunoassay.

Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field study in southern Vietnam, 155 cerebrospinal fluid (CSF) and 341 serum samples were collected from 111 children and 83 adults with suspected encephalitis. The JEV MAC DOT, performed on site, was scored visually from negative to strongly positive by two observers, and the results were compared subsequently with those of the standard IgM capture enzyme-linked immunosorbent assay. For the 179 patients with adequate specimens, the MAC DOT correctly identified 59 of 60 JEV-positive patients and 118 of 119 JEV-negative patients (sensitivity [95% confidence intervals], 98.3% [92.1 to 99.91%]; specificity, 99.2% [95.9 to 100.0%]; positive predictive value, 0.98; negative predictive value, 0.99). The MAC DOT also correctly identified three patients with dengue encephalopathy. Admission specimens were positive for 73% of JE patients. Interobserver agreement for MAC DOT diagnosis was excellent (kappa = 0.94). The JEV MAC DOT is a simple and reliable rapid diagnostic test for JE in rural hospitals.

Laboratory diagnosis of congenital syphilis by immunoglobulin M (IgM) and IgA immunoblotting.

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants.

Detection of Lassa virus antigens and Lassa virus-specific immunoglobulins G and M by enzyme-linked immunosorbent assay.

Rapid diagnosis of Lassa fever is desirable for the timely therapeutic intervention and implementation of strict quarantine procedures both in West Africa field hospitals where the disease is endemic and at international crossroads. An enzyme-linked immunosorbent assay (ELISA) to measure Lassa virus antigens in viremic sera was developed in which experimentally infected monkeys were used as a model for the human disease. In this test, Lassa virus antigens in test sera were captured in wells of microtiter plates by monkey anti-Lassa virus immunoglobulin. Guinea pig anti-Lassa virus immunoglobulin was then added, and binding of specific immunoglobulin was quantitated by the addition of rabbit anti-guinea pig immunoglobulin followed by alkaline phosphatase-labeled anti-rabbit immunoglobulin. This test detected viremia titers as low as 2.1 log10 PFU/ml in experimentally infected monkey sera, a titer often exceeded in patients with Lassa fever. Inactivation of infectious virus by beta-propiolactone or gamma-irradiation did not diminish reactivity. Antigen-ELISA concentrations increased with infectivity for the first 10 days after infection but then declined while infectivity titers remained high, suggesting that the presence of humoral antibody in viremic sera diminishes the sensitivity of the antigen ELISA. Lassa virus-specific immunoglobulin M (IgM) titers measured in an IgM capture ELISA were detectable within 10 days of infection and peaked after 36 days but remained detectable for 1.5 years. The Lassa virus-specific IgG ELISA response was slightly delayed, peaking on day 73 but declining only slightly thereafter. These studies in a realistic primate model suggest that the antigen detection ELISA or the IgM capture ELISA described, in which beta-propiolactone-inactivated sera are used, should be useful for the rapid diagnosis of human Lassa fever.