Early diagnosis with ultrasensitive ELISA.

Accurate, rapid and simple detection methods are required to facilitate early diagnosis of various disorders including infectious and lifestyle diseases as well as cancer. These detection approaches reduce the window of infection, i.e., the period between infection and reliable detection. Optimally, these methods should target protein as an indicator of pathogenic microbes as well as other biomarkers. For example, although nucleic acid is easily detected by polymerase chain reaction (PCR), these markers are also present in dead microbes, and, in the case of mRNA, it is not known whether this target was successfully translated. Accordingly, early diagnostic approaches require the development of ultrasensitive protein detection methods. In this chapter, we introduce an ultrasensitive enzyme-linked immunosorbent assay (ELISA) which combines a traditional sandwich-based immunoassay with thionicotinamide adenine dinucleotide (thio-NAD) cycling. The performance characteristics of this unique approach are reviewed as well as its potential role in providing a novel and ultrasensitive diagnostic tool in the clinical laboratory.

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity.

BACKGROUND: Nucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours. METHODS: A prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis. MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert). FINDINGS: The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10-19 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca. 330 CFU/mL in the culture method - almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 µg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0-92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9-93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli. INTERPRETATION: Our culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy. FUNDING: Matching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2.

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10-18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10-20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

Ultrasensitive enzyme-linked immunosorbent assay for the detection of MPT64 secretory antigen to evaluate Mycobacterium tuberculosis viability in sputum.

OBJECTIVES: This study examined Mycobacterium tuberculosis (MTB)-secreted MPT64 as a surrogate of bacterial viability for the diagnosis of active pulmonary TB (PTB) and for follow-up treatment. METHODS: In this proof-of-concept prospective study, 50 PTB patients in the Tokyo metropolitan region, between 2017 and 2018, were consecutively included and 30 healthy individuals were also included. Each PTB patient submitted sputum on days 0, 14 and 28 for diagnosis and follow-up, and each healthy individual submitted one sputum sample. The following were performed: smear microscopy, Xpert MTB/RIF, MGIT and solid culture, and MPT64 detection on the sputum samples. Ultrasensitive ELISA (usELISA) was used to detect MPT64. The receiver operating characteristic analyses for diagnosis and follow-up revealed the optimal cut-off value of MPT64 absorbance for detecting culture positivity at multiple intervals. RESULTS: The sensitivity of MPT64 for diagnosing PTB was 88.0% (95% CI 75.7-95.5) and the specificity was 96.7% (95% CI 82.8-99.9). The specificity of MPT64 for predicting negative culture results on day 14 was 89.5% (95% CI 66.9-98.7). The sensitivity of MPT64 for predicting positive culture results on day 28 was 81.0% (95% CI 58.1-94.6). CONCLUSIONS: This study revealed that MPT64 is useful for diagnosing active PTB in patients and predicting treatment efficacy at follow-up.

Ultrasensitive ELISA Developed for Diagnosis.

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of a sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 405 nm. We have successfully achieved a limit of detection of ca. 10-18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10-18 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10-19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy.

Objective: The chronic kidney disease (CKD) is widely diagnosed on the basis of albuminuria and the glomerular filtration rate. A more precise diagnosis of CKD, however, requires the assessment of other factors. Urinary adiponectin recently attracted attention for CKD assessment, but evaluation is difficult due to the very low concentration of urinary adiponectin in normal subjects. Research design and methods: We developed an ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide cycling to detect trace amounts of proteins, which allows us to measure urinary adiponectin at the subattomole level. We measured urinary adiponectin levels in 59 patients with diabetes mellitus (DM) and 24 subjects without DM (normal) to test our hypothesis that urinary adiponectin levels increase with progression of CKD due to DM. Results: The urinary adiponectin levels were 14.88±3.16 (ng/mg creatinine, mean±SEM) for patients with DM, and 3.06±0.33 (ng/mg creatinine) for normal subjects. The threshold between them was 4.0 ng/mg creatinine. The urinary adiponectin levels increased with an increase in the CKD risk. Furthermore, urinary adiponectin mainly formed a medium-molecular weight multimer (a hexamer) in patients with DM, whereas it formed only a low-molecular weight multimer (a trimer) in normal subjects. That is, the increase in urinary adiponectin in patients with DM led to the emergence of a medium-molecular weight form in urine. Conclusions: Our new assay showed that urinary adiponectin could be a new diagnostic index for CKD. This assay is a non-invasive test using only urine, thus reducing the patient burden.

Immunochromatographic detection of MPB64 secreted from active BCG by heating: toward same-day diagnosis of tuberculosis.

We propose a new detection method for a tuberculosis-specific protein, MPB64, obtained from active bacillus Calmette-Guérin (BCG) by heating. When BCG was included in solution at a concentration >2.75 × 104 CFU/ml, our method for collecting MPB64 through heating active BCG combined with an immunochromatographic assay detected active bacilli within 2.5 h. By contrast, a culture test, which is the gold standard for tuberculosis diagnosis, does not provide results for between 1 week and 2 months. The rapid tests based on PCR have some drawbacks, for example they detect DNA from both active and latent (or even dead) tubercle bacilli. Therefore, our method may pave the way toward detecting only active tubercle bacilli at a reasonable cost and providing same-day diagnosis.

Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus.

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016‒2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.

Development of an Influenza Rapid Diagnostic Kit Specific for the H7 Subtype.

Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients.

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.

Ultrasensitive detection of proteins and sugars at single-cell level.

Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine.

Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling.

To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 µL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10(-18) moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10(-18) moles of the p24/assay corresponds to ca. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling.

Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10(-19) moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method.

An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10(-19) mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.