Antibody Immunoassay Research Articles

Posterior reversible encephalopathy syndrome in a patient with a metastatic pancreatic neuroendocrine tumour and ectopic Cushing's syndrome.

SummaryWe report a rare case of posterior reversible encephalopathy syndrome (PRES), precipitated by ectopic Cushing’s syndrome, in a patient with a metastatic pancreatic neuroendocrine tumour. A 55-year-old female presented as a hypertensive emergency with seizures and severe biochemical disturbance, including alkalosis, hypokalaemia and hyperglycaemia. MRI showed vasogenic oedema in the parieto-occipital region, consistent with a diagnosis of PRES. She had a significantly raised serum cortisol (>6000 nmol/L) which did not suppress with dexamethasone. Plasma adrenocorticotropic hormone (ACTH) concentrations were neither suppressed nor raised but were consistently within the normal reference range. The unexpected finding of a normal ACTH may be explained by either tumour secretion of unmeasured ACTH-related peptides, immunoassay antibody interference or episodic ACTH secretion. PRES is usually reversible with prompt and appropriate treatment. Hypercortisolism associated PRES is rare and may be associated with a worse outcome.Learning pointsPRES secondary to ectopic Cushing’s syndrome is very rare.PRES in this context may indicate a worse prognosis.In ectopic Cushing’s syndrome, if the serum ACTH level is normal, consider testing for ACTH-related peptides or interfering antibodies.Further research is required to establish the best treatment approach and to improve patients’ outcomes.

Hapten synthesis, monoclonal antibody production and immunoassay development for direct detection of 4-hydroxybenzehydrazide in chicken, the metabolite of nifuroxazide

Derivatization is usually employed in immunoassay for detection of metabolites of nitrofurans and avoiding derivatization could be preferable to achieve an efficient screening. In the study, we designed four haptens of 4-hydroxybenhydrazide (HBH), the nifuroxazide metabolite. The effect of hapten structures on antibody affinity were evaluated and one monoclonal antibody was produced by using the Hapten C with a linear alkalane spacer arm. After optimization, an enzyme linked-immunosorbent assay (ELISA) was established with an 50% inhibition concentration of 0.25 ng mL−1 for HBH, which could ensure the direct detection of HBH without derivatization. The limit of detection of the ELISA for HBH was 0.12 µg kg−1 with the recoveries of 90.1–96.2% and coefficient of variation (CV) values lower than 9.1%. In conclusion, we produced several high affinity antibodies to HBH with new designed hapten and developed an icELISA for the direct detection of HBH without derivatization in chicken.

Accurate point-of-care serology tests for COVID-19.

As COVID-19 vaccines become available, screening individuals for prior COVID-19 infection and vaccine response in point-of-care (POC) settings has renewed interest. We prospectively screened at-risk individuals for SARS-CoV-2 spike and nucleocapsid protein antibodies in a POC setting to determine if it was a feasible method to identify antibody from prior infection. Three EUA-approved lateral flow antibody assays were performed on POC finger-stick blood and compared with serum and a CLIA nucleocapsid antibody immunoassay. Variables including antibody class, time since PCR, and the assay antigen used were evaluated. 512 subjects enrolled, of which 104 had a COVID-19 history and positive PCR. Only three PCR-positive subjects required hospitalization, with one requiring mechanical ventilation. The POC results correlated well with the immunoassay (93-97% sensitivity) and using serum did not improve the sensitivity or specificity. Finger-stick, POC COVID-19 antibody testing was highly effective in identifying antibody resulting from prior infections in mildly symptomatic subjects. Using high-complexity serum immunoassays did not improve the screening outcome. Almost all individuals with COVID-19 infection produced detectable antibodies to the virus. POC antibody testing is useful as a screen for prior COVID-19 infection, and should be useful in assessing vaccine response.

Prevalence of heterophilic antibodies in serum samples from horses in an equine hospital, and elimination of interference using chicken IgY

BackgroundHeterophilic antibodies in serum and plasma can interfere with mammalian antibodies in immunoassays and result in false test results, usually false positive. Although studies screening for heterophilic antibodies as well as elimination studies have been conducted in dogs and cats, knowledge of the presence of heterophilic antibodies in other species in veterinary medicine is limited. In this study, a 2-site sandwich-type interference assay that detects anti-mouse antibodies was used to detect heterophilic antibodies in a population of horses treated in an animal hospital.ResultsA total of 194 serum samples from 127 individual horses were analyzed. There were 11/127 (8.7%) interference-positive horses, and these were analyzed in an assay exchanging the capture mouse IgG with chicken IgY. The positive samples were negative in the chicken IgY assay, indicating elimination of a possible interference, with the chicken-based assay. Four interference-positive samples were from geldings, and anti-Müllerian hormone (AMH) was analyzed from these samples. AMH concentrations were negative in these samples as expected in geldings, indicating that the heterophilic antibodies did not cause interference in the AMH assay.ConclusionThe present study shows that there are heterophilic antibodies in horse serum samples like in samples from humans, dogs, and cats. The use of chicken-based reagents, such as chicken IgY, which do not cross-react with mammalian IgG, eliminates the effects of interfering antibodies in the samples. Equine heterophilic antibodies do not necessarily cause interference in commercial immunoassays.

MRNA SARS-CoV-2 Immunization Confers Robust Antibody Response in Occupational Healthcare Workers and Fosters Workplace Safety.

mRNA SARS-CoV-2 Immunization Confers Robust Antibody Response in Occupational Healthcare Workers and Fosters Workplace Safety.

New Cutoffs for the Biochemical Diagnosis of Adrenal Insufficiency after ACTH Stimulation using Specific Cortisol Assays.

ContextThe normal cortisol response 30 or 60 minutes after cosyntropin (ACTH[1–24]) is considered to be ≥18 μg/dL (500 nmol/L). This threshold is based on older serum cortisol assays. Specific monoclonal antibody immunoassays or LC-MS/MS may have lower thresholds for a normal response.ObjectiveTo calculate serum cortisol cutoff values for adrenocorticotropic hormone (ACTH) stimulation testing with newer specific cortisol assays.MethodsRetrospective analysis of ACTH stimulation tests performed in ambulatory and hospitalized patients suspected of adrenal insufficiency (AI). Serum samples were assayed for cortisol in parallel using Elecsys I and Elecsys II immunoassays, and when volume was available, by Access immunoassay and LC-MS/MS.ResultsA total of 110 patients were evaluated. Using 18 μg/dL as the cortisol cutoff after ACTH stimulation, 14.5%, 29%, 22.4%, and 32% of patients had a biochemical diagnosis of AI using the Elecsys I, Elecsys II, Access, and LC-MS/MS assays, respectively. Deming regressions of serum cortisol were used to calculate new cortisol cutoffs based on the Elecsys I cutoff of 18 μg/dL. For 30-minute values, new cutoffs were 14.6 μg/dL for Elecsys II, 14.8 μg/dL for Access, and 14.5 μg/dL for LC-MS/MS. Baseline cortisol <2 μg/dL was predictive of subnormal stimulated cortisol values.ConclusionTo reduce false positive ACTH stimulation testing, we recommend a new serum cortisol cutoff of 14 to 15 μg/dL depending on the assay used (instead of the historical value of 18 μg/dL with older polyclonal antibody assays). Clinicians should be aware of the new cutoffs for the assays available to them when evaluating patients for AI.

The performance of hepatitis C virus (HCV) antibody point-of-care tests on oral fluid or whole blood and dried blood spot testing for HCV serology and viral load among individuals at higher risk for HCV in South Africa.

Background and AimsTo enhance screening and diagnosis in those at‐risk of hepatitis C virus (HCV), efficient and improved sampling and testing is required. We investigated the performance of point‐of‐care (POC) tests and dried blood spots (DBS) for HCV antibody and HCV RNA quantification in individuals at higher risk for HCV (people who use and inject drugs, sex workers and men who have sex with men) in seven South African cities.MethodsSamples were screened on the OraQuick HCV POC test (471 whole blood and 218 oral fluid); 218 whole blood and DBS paired samples were evaluated on the ARCHITECT HCV antibody (Abbott) and HCV viral load (COBAS Ampliprep/COBAS TaqMan version 2) assays. For HCV RNA quantification, 107 dB were analyzed with and without normalization coefficients.ResultsPOC on either whole blood or oral fluid showed an overall sensitivity of 98.5% (95% CI 97.4‐99.5), specificity of 98.2% (95% CI 98.8‐100) and accuracy of 98.4% (95% CI 96.5‐99.3). On the antibody immunoassay, DBS showed a sensitivity of 96.0% (95% CI 93.4‐98.6), specificity of 97% (95% CI 94.8‐99.3) and accuracy of 96.3% (95% CI 93.8‐98.8). A strong correlation (R 2 = 0.90) between viral load measurements for DBS and plasma samples was observed. After normalization, DBS viral load results showed an improved bias from 0.5 to 0.16 log10 IU/mL.ConclusionThe POC test performed sufficiently well to be used for HCV screening in at‐risk populations. DBS for diagnosis and quantification was accurate and should be considered as an alternative sample to test. POC and DBS can help scale up hepatitis services in the country, in light of our elimination goals.

COVID-19 Antibody Tests and Their Limitations.

COVID-19, caused by the SARS-CoV-2 virus, has developed into a global health crisis, causing over 2 million deaths and changing people’s daily life the world over. Current main-stream diagnostic methods in the laboratory include nucleic acid PCR tests and direct viral antigen tests for detecting active infections, and indirect human antibody tests specific to SARS-CoV-2 to detect prior exposure. In this Perspective, we briefly describe the PCR and antigen tests and then focus mainly on existing antibody tests and their limitations including inaccuracies and possible causes of unreliability. False negatives in antibody immunoassays can arise from assay formats, selection of viral antigens and antibody types, diagnostic testing windows, individual variance, and fluctuation in antibody levels. Reasons for false positives in antibody immunoassays mainly involve antibody cross-reactivity from other viruses, as well as autoimmune disease. The spectrum bias has an effect on both the false negatives and false positives. For assay developers, not only improvement of assay formats but also selection of viral antigens and isotopes of human antibodies need to be carefully considered to improve sensitivity and specificity. For clinicians, the factors influencing the accuracy of assays must be kept in mind to test patients using currently imperfect but available tests with smart tactics and realistic interpretation of the test results.

Nuclear magnetic resonance immunoassay of tetanus antibodies based on the displacement of magnetic nanoparticles.

A nuclear magnetic resonance (NMR) immunoassay based on the application of carbon-coated iron nanoparticles conjugated with recognition molecules was designed. The principle of the assay is that ELISA plates are coated with a capture element, and then an analyte is added and detected by conjugating the magnetic nanoparticles with recognition molecules. Afterwards, the elution solution (0.1-M sodium hydroxide) is added to displace the magnetic nanoparticles from the well surfaces into the solution. The detached magnetic nanoparticles reduce transverse relaxation time (T2) values of protons from the surrounding solution. A portable NMR relaxometer is used to measure the T2. Magnetic nanoparticles conjugated with streptavidin, monoclonal antibodies, and protein G were applied for the detection of biotinylated albumin, prostate-specific antigen, and IgG specific to tetanus toxoid (TT). The limit of detection of anti-TT IgG was 0.08-0.12 mIU/mL. The reproducibility of the assay was within the acceptable range (CV < 7.4%). The key novelty of the immunoassay is that the displacement of the nanoparticles from the solid support by the elution solution allows the advantages of the solid phase assay to be combined with the sensitive detection of the T2 changes in a volume of liquid.

Evaluation of four laboratory-based SARS-CoV-2 IgG antibody immunoassays

ObjectivesTo evaluate the diagnostic performances of four SARS-CoV-2 IgG antibody immunoassays. MethodsFollowing immunoassays were studied: Abbott's SARS-CoV-2 IgG assay, Diasorin's Liaison SARS-CoV-2 S2/S2 IgG assay, Euroimmun's Anti-SARS-CoV-2 IgG ELISA, and Roche's Elecsys Anti-SARS-CoV-2 assay. Specificity was retrospectively evaluated with 38 samples from 2019. Sensitivity samples (n = 147) were taken from SARS-CoV-2 real-time PCR-positive patients who developed COVID-19 symptoms ten days earlier. ResultsMean specificity was 96.6%. Mean sensitivity was 62.7% from ten days after onset of symptoms, 84.4% from 15 days after onset of symptoms, and 87.5% from 20 days after onset of symptoms. ConclusionsSpecificity was high, while Abbott and Roche were 100% specific. Sensitivity increased over time, with Abbott and Roche having the highest sensitivity at all time points with ≥90% from 20 days after symptoms' onset. These findings may assist in selecting SARS-CoV-2 IgG antibody immunoassays for additional diagnostics, epidemiological research, and vaccine development.

Orthogonal immunoassays for IgG antibodies to SARS-CoV-2 antigens reveal that immune response lasts beyond 4 mo post illness onset.

Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the current pandemic remains a field of immense interest and active research worldwide. Although the severity of acute infection may depend on the intensity of innate and adaptive immunity, leading to higher morbidity and mortality, the longevity of IgG antibodies, including neutralizing activity to SARS-CoV-2, is viewed as a key correlate of immune protection. Amid reports and concern that there is a rapid decay of IgG antibody levels within 1 mo to 2 mo after acute infection, we set out to study the pattern and duration of IgG antibody response to various SARS-CoV-2 antigens in asymptomatic and symptomatic patients in a community setting. Herein, we show the correlation of IgG anti-spike protein S1 subunit, receptor binding domain, nucleocapsid, and virus neutralizing antibody titers with each other and with clinical features such as length and severity of COVID-19 illness. More importantly, using orthogonal measurements, we found the IgG titers to persist for more than 4 mo post symptom onset, implying that long-lasting immunity to COVID-19 from infection or vaccination might be observed, as seen with other coronaviruses such as SARS and Middle East respiratory syndrome.

Improved Diagnosis of Tuberculous Meningitis Using a Combination of Multiplex Antigens and Antibodies Testing Methods

Introduction: Various serological assays exists, including Antigen and Antibody detection (IgM and IgG) for diagnosis of Tuberculous Meningitis (TBM) cases. To the best of our knowledge, most of the laboratories either do Antigen detection or IgM or IgG, at a time. As different antigens get expressed in a different stage of infection it may be possible that many cases remain undiagnosed due to one test at a time approach. Aim: To evaluate the combination of Mycobacterium Tuberculosis (MTB) antigen (Ag85 Complex and Rv2623) and antibody (Anti-Ag85, Anti-45kD, Anti-HSP-16, Anti-CFP-10 Anti-GroES and Anti-ESAT-6) immunoassay panels in the Cerebrospinal Fluid (CSF) samples for diagnosis of TBM patient. Materials and Methods: In the present prospective study conducted at Central India Institute of Medical Sciences (CIIMS) from October 2013 to April 2015, a total of 200 CSF samples of different groups {confirmed TBM (n=100) and noninfectious neurological diseases as control (n=100)} were analysed by Enzyme-Linked Immunosorbent Assay (ELISA). A panel of MTB antigens consisting of Ag85B, 45kDa, HSP-16, CFP-10, GroES and ESAT-6 were used for detection of antibodies response, whereas polyclonal antibodies were used for antigen detection of Ag85 complex and Rv2623 in the CSF samples. The comparison of the CSF parameter between TBM and non-TBM patients was performed using a student t-test. A p-value &lt;0.05 was considered statistically significant for all the analyses. Results: The study population has similar age and sex distribution (p&gt;0.05). Symptoms of headache, fever, neck stiffness, vomiting, abnormal behaviour, unconsciousness were more common among the TBM patients as compared to non-TBM patients (p&lt;0.05) (TBM Vs non-TBM). Similarly TBM patients had an increase (p&lt;0.05) Vs non-TBM total cell count, protein, parallel blood sugar and decline in CSF sugar and Parallel blood sugar ratio (p&lt;0.05) (TBM Vs non-TBM). We found diagnostic accuracy of 67% to 76% with either antigen or antibody assay, however, combinations of antigen and antibody immunoassay together increase the diagnostic accuracy of up to 96%. Conclusion: Our study recommends that a combination of antigen and antibody assay should be considered for early and accurate diagnosis of TBM cases.

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics, Content and Time Course of SARS-CoV-2 Neutralizing Antibodies

Background: Convalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma. Methods: Patients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed. Results: A series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from <1:20 to >1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels. Conclusion: We demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.

A Rapid and Highly Sensitive Chemiluminescent Method to Quantify HPV16E7 Antibody Based on Immunomagnetic Separation Methods.

In order to detect anti-HPV16E7 antibody in serum, a highly sensitive and rapid detection method based on chemiluminescence immunoassay and immunomagnetic separation was introduced. The technique that was developed is a novel, sensitive chemiluminescence (CL) immunoassay for HPV16E7 antibody detection. Balb/C mice were immunized with HPV16E7 fusion protein to prepare monoclonal antibody against HPV16E7. The biotinylated antigen was prepared as immunomagnetic beads and its stability was tested (IMBs). The protocol used horseradish peroxidase (HRP) labeled HPV16E7 antigen and immunomagnetic beads (IMBs). The antibody induced the formation of IMBs-mAbs HPV16E7-HRP labeled antibody structures. IMBs were applied to capture CEA and immobilize CEA through the external magnetic field. Oxidized luminescence substrate can be catalyzed by HRP on antibody surface to generate optical signals which were detected by luminometer. HPV16E7 monoclonal antibody was prepared and validated. The HPV16E7 antigen can efficiently bind to the bead with a conjugation rate of 72%. The biological activity of IMBs did not decrease significantly when stored in the dark at 4℃ for 2 months. The sensitivity and stability of this proposed method were excellent and showed a good linear relationship (Y = 1.3203 X + 0.7, R2 = 0.9952). This proposed technique showed excellent performance in quantitative measurement of HPV6E7 and was expected to be used in clinical detection.

Validation of dried blood spot sample modifications to two commercially available COVID-19 IgG antibody immunoassays.

Aim: Coronavirus disease 2019 antibody testing often relies on venous blood collection, which is labor-intensive, inconvenient and expensive compared with finger-stick capillary dried blood spot (DBS) collection. The purpose of our work was to determine if two commercially available anti-severe acute respiratory syndrome coronavirus 2 enzyme-linked immunosorbent assays for IgG antibodies against spike S1 subunit and nucleocapsid proteins could be validated for use with DBS. Materials & methods: Kit supplied reagents were used to extract DBS, and in-house DBS calibrators were included on every run. Results: Positive/negative concordance between DBS and serum was 100/99.3% for the spike S1 subunit assay and 100/98% for the nucleocapsid assay. Conclusion: Validation of the DBS Coronavirus disease 2019 IgG antibody assays demonstrated that serum and DBS can produce equivalent results with minimal kit modifications.

Infection fatality risk for SARS-CoV-2 in community dwelling population of Spain: nationwide seroepidemiological study

ObjectiveTo estimate the infection fatality risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on deaths with confirmed coronavirus disease 2019 (covid-19) and excess deaths from all causes.DesignNationwide seroepidemiological...

Evaluation of Humoral Immunity to SARS-CoV-2: Diagnostic Value of a New Multiplex Addressable Laser Bead Immunoassay.

Despite efforts to develop anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Ab) immunoassays, reliable serological methods are still needed. We developed a multiplex addressable laser bead immunoassay (ALBIA) to detect and quantify anti-Spike S1 and nucleocapsid N Abs. Recombinant S1 and N proteins were bound to fluorescent beads (ALBIA-IgG-S1/N). Abs were revealed using class-specific anti-human Ig Abs. The performances of the test were analyzed on 575 serum samples including 192 from SARS-CoV-2 polymerase chain reaction–confirmed patients, 13 from seasonal coronaviruses, 70 from different inflammatory/autoimmune diseases, and 300 from healthy donors. Anti-S1 IgM were detected by monoplex ALBIA-IgM-S1. Comparison with chemiluminescent assays or enzyme-linked immunosorbent assays was performed using commercial tests. Multiplex ALBIA-IgG-S1/N was effective in detecting and quantifying anti–SARS-CoV-2 IgG Abs. Two weeks after first symptoms, sensitivity and specificity were 97.7 and 98.0% (anti-S1), and 100 and 98.7% (anti-N), respectively. Agreement with commercial tests was good to excellent, with a higher sensitivity of ALBIA. ALBIA-IgG-S1/N was positive in 53% of patients up to day 7, and in 75% between days 7 and 13. For ALBIA-IgM-S1, sensitivity and specificity were 74.4 and 98.7%, respectively. Patients in intensive care units had higher IgG Ab levels (Mann–Whitney test, p < 0.05). ALBIA provides a robust method for exploring humoral immunity to SARS-CoV-2. Serology should be performed after 2 weeks following first symptoms, when all COVID-19 (coronavirus disease 2019) patients had at least one anti-S1 or anti-N IgG Ab, illustrating the interest of a multiplex test.

Seroprevalence of Antibodies Against SARS-CoV-2 Among Health Care Workers in Mumbai, India.

To ascertain the seroprevalence of antibodies against SARS-CoV-2 among health care workers in tertiary care hospitals in Mumbai, India. Health care workers (801) from designated COVID-19 hospitals (400) and non-COVID-19 facilities (401) underwent an electrochemiluminescent automated immunoassay for antibodies to SARS-CoV-2. Details including demographics, comorbidities, symptoms compatible with COVID-19, contact with COVID-19 individuals, personal protective equipment use at work, and details of polymerase chain reaction tests were collected through a validated questionnaire. Doctors (201, 25.1%), nurses (308, 38.5%), and ancillary workers (292, 36.5%) participated in the study. Seroprevalence in 801 participants was 11.1% (9.1% to 13.5%). It was significantly higher ancillary workers (18.5%, 14.5% to 23.3%) than doctors (7%, 4.2% to 11.4%) and nurses (6.8%, 4.5% to 10.2%). Seroprevalence was significantly higher in non-COVID-19 hospitals (13.5%, 10.5% to 17.2%) than COVID-19 hospitals (8.7%, 6.3% to 11.9%). Having a COVID-19 household contact was a significant risk for seropositivity (18.9% vs 10.3%), while a neighborhood contact did not affect seropositivity (9.4% vs 7.3%). Loss of taste/smell and fever were only 2 symptoms associated with seropositivity. Comorbidities did not affect the seropositivity rate. Seropositivity was likely to be higher in ancillary workers and in non-COVID hospitals. There is need to enhance COVID protection protocols and awareness among all health care workers.

Wide-Detectable Range Electrochemical Immunosensor in PPQ to PPM

In this research, we propose the concept of electrochemical immunosensing. The typical antibody had a difficulty to detect a small analyte. In conventional method, the Fab region of antibody is extracted after the decomposition of antibody, however it is still difficult to apply in the detection of tiny analyte. In this research, we propose here a use of only a biorecognition element of antibody for immunoassay. In recent, our research group developed the synthesis method of variable domain ofheavy chain of heavy-chain antibody (VHH antibody) from Brevibacillus with enzyme. VHH antibody is a recognitionpart of antibody. Thus, it is physically and chemically stable, and heat-resist characteristic.Here, the electrochemical immunosensing of ovalbumin (ca. 14 nm) is demonstrated. OVA VHH antibody was newly synthesized for this research.The sensor chip is updated into planar-sensor chip. Four electrodes are assigned forming ring on the chip surface. All the substrate of electrodes were made from Au on Cu. The whole size of this sensor chip is 105 mm x 11 mm. Both counter electrode and reference electrode are Au.Working electrode was mixed monolayer of VHH antibody-propanethiol/Au, ferrocenyl-propanethiol/Au, and ethanolic-propanethiol/Au. Ethanolic-propanethiol was used for prevention of non-specific adsorption of protein.Ferricyanide plays a role in the promoter for redox reaction of ferrocene.Once the electrochemical immunosensor chip was contacted with OVA standard solution (1 ppt to 10 ppm of OVA in PBS solution), and then the chip was rinsed with PBS solution. And then, cyclic voltammogram was obtained in 0.1 M NaCl electrolyte solution. For further amplification of the signal, ferricyanide was added to the electrolyte solution. Figure 1 illustrates cyclic voltammograms of electrochemical immunosensor after dumping with OVA standard solution in the range from 1 ppt to 10 ppm. The OVA VHH antibody captured OVA in the process of antigen- antibody reaction. As a result, captured OVA blocked the redox reaction of amplifier. It can be observed in the Figure 1 as a delay and decay of the peak. Regeneration of the surface of the sensor chip was completed except for yellow line (100 ppt, Figure 1).It means that when the regeneration succeeded, we can make use of this sensor chip again in another measurement.Two kind of calibration plots were obtained from the current density at +0.35 V and the peak potential in Figure 1.It can distinguish from each peak from 1 ppt to 10 ppm. It means to be able to detect in the broad range.Another plot of the anodic current at +0.35 V showed that the OVA of 0 to 10 ppt could be very sensitively detected.It is critical that this sensor chip is valuable because of its sensitivity, repeatability, reproducibility, and compact. It can be presumed that it can apply to real-time sensing or detection of ultra- micro substance in the organisms.

First case of molecularly identified and genetically characterized human T-cell leukemia virus type 2 infection in a pregnant woman in non-endemic Japan

Human T-cell leukemia virus type 2 (HTLV-2) is non-endemic in Japan unlike the related HTLV type 1. Previously, although HTLV-2-seropositivity was identified via western blotting in one male blood donor in Japan, there have been no reports of HTLV-2 provirus detection by nucleic acid testing. In this report, one Japanese pregnant woman was clinically diagnosed as being HTLV-2-infected with a line immunoassay for specific antibodies after primary testing through prenatal screening in Japan. In genomic DNA of her peripheral blood mononuclear cells, HTLV-2 proviral genome was detected by nucleic acid testing (three methods) with quantitative polymerase chain reaction. The full-genome sequence of this strain was successfully determined. The identified virus was interestingly characterized as a presumed progenitor of subtypes a and c by recombination region and phylogenetic tree analyses. In conclusion, the present infection is, to our knowledge, the first case of molecularly identified and genetically characterized HTLV-2 infection found via prenatal screening in non-endemic Japan.