Two-step Enzyme-linked Immunosorbent Assay Research Articles

Detection frequency and duration of circulating antiphospholipid syndrome markers in patients with verified COVID-19

Aim: to develop enzyme-linked immunosorbent tests for assessing the antiphospholipid syndrome (APS) markers and determine prevalence of three antiphospholipid antibody (aPL) types at different COVID-19 stages. Materials and Methods. A comparative longitudinal controlled study was conducted by examining 120 subjects with COVID-19 diagnosis verified by reverse transcription polymerase chain reaction. Donor serum samples collected before November 2019 were used as a control group. The laboratory study included measurement of IgA, IgM and IgG against β2-glycoprotein 1 (β2-GP1), cardiolipin, phosphatidylserine-prothrombin complex (PS-PT) by using domestically produced test systems based on indirect two-step enzyme-linked immunosorbent assay. Results. Validation of the developed experimental tests was carried out in comparison with foreign commercial analogues in accordance with international standards. Alternative antigenic targets for effective diagnosis of antibodies against β2-GP1 were studied. Analyzing rate of aPL in patients at different COVID-19 stages showed that in acute vs. convalescence stage it was higher by 1.3-fold (81.7 and 65.0 %, respectively). The first rank detection place was assigned to IgG against β2-GP1, cardiolipin and PS-PT, the second – IgM against cardiolipin. The profile of the detected antibodies changed at various COVID-19 stages driven by time frame elapsed from the moment of diagnosis. Conclusion. Recombinant constructs are created and analytical conditions are optimized for determining various aPL types. It was shown that along with other viral infections, COVID-19 triggers autoantibody production demonstrating that 54.2 % individuals infected with SARS-CoV-2 were positive at least for one autoantibody type. The majority of such virus-associated aPL are presumably transiently positive.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Serum Bioavailable, Rather Than Total, 25-hydroxyvitamin D Levels Are Associated With Hepatocellular Carcinoma Survival.

Background and AimsFree and bioavailable 25‐hydroxyvitamin D (25OHD) are emerging measurements of vitamin D status. It remains unclear whether circulating free or bioavailable 25OHD are relevant to hepatocellular carcinoma (HCC) prognosis. Our aim was to test the hypothesis that bioavailable 25OHD may be a better serum biomarker of vitamin D status than total 25OHD on the association with HCC survival.Approach and ResultsWe included 1,031 newly diagnosed, previously untreated patients with HCC from the Guangdong Liver Cancer Cohort enrolled between September 2013 and April 2017. Serum total 25OHD levels were measured using an electrochemiluminescence immunoassay. Serum‐free 25OHD levels were measured using a two‐step enzyme‐linked immunosorbent assay. Bioavailable 25OHD levels were calculated from measured free 25OHD and albumin using a previously validated equation. Primary outcomes were liver cancer–specific (LCSS) and overall survival (OS). Cox proportional hazards models were performed to calculate the multivariable hazard ratios (HRs) and 95% confidence intervals (CIs). During a median follow‐up of 726 days, 430 patients had deceased, including 393 deaths from HCC. In multivariable analyses, higher bioavailable 25OHD levels were significantly associated with better survival, independent of nonclinical and clinical prognostic factors including serum C‐reactive protein, Barcelona Clinic Liver Cancer stage, and cancer treatment. The multivariable‐adjusted HRs in the highest versus lowest quartile of bioavailable 25OHD levels were 0.69 (95% CI: 0.51, 0.93; P for trend = 0.014) for LCSS and 0.71 (95% CI: 0.53, 0.94; P for trend = 0.013) for OS. In contrast, neither total nor free 25OHD levels were associated with LCSS or OS.ConclusionsHigher bioavailable, rather than total, 25OHD levels were independently associated with improved survival in a population‐based HCC cohort, suggesting a potential utility of bioavailable 25OHD in HCC prognosis.

Anti-JC Virus Antibody Prevalence in Canadian MS Patients.

Anti-John Cunningham (JCV) antibodies have been detected in approximately 50% to 60% of multiple sclerosis (MS) patients. Age, sex, and geographic location have been associated with seroprevalence differences. We describe anti-JCV antibody prevalence in the Canadian cohort of patients enrolled in the JCV Epidemiology in MS study. This cross-sectional multicenter study evaluated the effects of demographic and disease characteristics on anti-JCV antibody seroprevalence in MS patients irrespective of disease type and treatment. A single blood sample was collected for analysis of anti-JCV antibodies using a two-step enzyme-linked immunosorbent assay (ELISA). Chi-square and logistic regression tests were used to determine significance. A total of 4198 Canadian MS patients participated in the study; the overall anti-JCV antibody prevalence was 56.3% (95% confidence interval: 54.8% to 57.8%). Seroprevalence was significantly associated with age (increasing from 45% in young to 61% in those >60 years), sex, and region (p<0.0001 for age and sex; p=0.005 for region). No significant differences in anti-JCV antibody prevalence were associated with race, MS disease type and duration, or number and duration of treatments. Immunosuppressant use was associated with a higher seroprevalence rate (63.4%) compared with no immunosuppressant use (55.9%; p=0.040). Canadian MS patients had an overall anti-JCV antibody seroprevalence that was consistent with previous studies using the two-step ELISA. Significant associations of anti-JCV antibody positivity were found with age, sex, region, and immunosuppressant therapy, whereas seroprevalence was not associated with race, MS type, MS duration, or number or duration of MS treatments.

JC virus antibody index in natalizumab-treated patients: correlations with John Cunningham virus DNA and C-reactive protein level.

Natalizumab-treated patients have a higher risk of developing progressive multifocal leukoencephalopathy. Exposure to John Cunningham virus (JCV) is a prerequisite for PML (progressive multifocal leukoencephalopathy). To assess JCV exposure in multiple sclerosis patients, we performed a serological examination, obtained the antibody index, performed real-time polymerase chain reaction (PCR) to detect JCV DNA in plasma and urine, and investigated the role of ultrasensitive C-reactive protein (usCRP) as a possible biological marker of JCV reactivation. We retrospectively analyzed consecutive natalizumab-treated multiple sclerosis patients who underwent a JCV antibody test through a two-step enzyme-linked immunosorbent assay (STRATIFY test) to the measure of serum usCRP levels, and to perform blood and urine JCV PCR. The studied cohort included 97 relapsing–remitting patients (60 women). Fifty-two patients (53.6%) tested positive for anti-JCV antibodies. PCR showed JCV DNA in the urine of 30 out of 83 (36.1%) patients and 28 out of 44 seropositive patients (63.6%), with a 6.7% false-negative rate for the STRATIFY test. Normalized optical density values were higher in urinary JCV DNA-positive patients (P<0.0001). Interestingly, the level of usCRP was higher in urinary JCV DNA-positive patients and correlated to the number of DNA copies in urine (P=0.028). As expected, patients’ age correlated with JCV seropositivity and with JC viruria (P=0.02 and P=0.001, respectively). JC viruria was significantly correlated with a high JCV antibody index and high serum usCRP levels. We suggest that PCR and usCRP might be useful as markers of JCV reactivation, and that patients should be monitored between STRATIFY assessments.

Negative Interference in Serum HBsAg ELISA from Rheumatoid Factors

BackgroundRF(Rheumatoid factor) is usually thought to cause positive interference in immunoassay. Recently, our study showed that high-concentration RFs caused negative interference as well as positive interference in serum HBsAg(Hepatitis B surface antigen) ELISA(Enzyme-linked immunosorbent assay), but it is unclear that RF causing negative interference is an anomaly produced by a certain ELISA kit or a common property of most of HBsAg ELISA kits.MethodsSerum models were made by blending HBsAg-positive sera and high- or moderate-concentration RFs sera at the ratio of 1: 9, then one-step and two-step ELISA were adopted to determine HBsAg in serum models.ResultsNo matter what kind of kit used, one-step ELISA showed that HBsAg S/CO( sample/cut off) values in serum models were significantly lower than original values. Bivariate correlations tests showed decline rates of HBsAg S/CO Values were not associated to serum RF concentrations ranging from 288 to 3560 IU/mL. HBsAg converted to be negative in 69.80% serum models with original-value ranging from 1.00 to 10.00, and in 2.68% serum models with higher original-value. RF causing decline of HBsAg S/CO value provided by one-step ELISA was more obvious than that provided by two-step ELISA. ConclusionsIt is concluded that susceptibility of all HBsAg ELISA assays to interference from RF, leading to predominantly lower and in some cases "false-negative" results, and moreover, the lower the original HBsAg S/CO Value, the higher the false-negative rate.

Diagnostic Value Of Serum Level Of Soluble CD14-Subtype In Febrile Neutropenia In Patients With Hematologic Disorders

Background Febrile neutropenia (FN) is a common and potentially fatal complication that can occur during intensive chemotherapy in patients with hematologic disorders. Identification of the cause of FN is very difficult due to the poor diagnostic performance of blood cultures. Thus, the standard of care for FN has been a fever-driven approach with the use of broad-spectrum antibiotics, likely resulting in over-treatment, poor cost-effectiveness, and induction of drug-resistance in bacteria. A novel diagnostic method is needed to improve these problems. CD14, one of the surface markers in monocytes/macrophages, is a lipopolysaccharide-binding protein complex receptor. Some recent studies reported that a fragment released into the blood—soluble CD14-subtype (sCD14-ST)—is promising as a new diagnostic biomarker of infection, especially sepsis (Endo S, et al. J Infect Chemother. 2012;18:891-7). However, the utility of this test in FN is unknown. Methods We prospectively examined the diagnostic value of serum sCD14-ST in patients with hematological disorders who developed FN after chemotherapy or hematopoietic cell transplantation between November 2010 and February 2012. Neutropenia was defined as a neutrophil count of less than 500/μL or less than 1,000/μL with an expected decline to less than 500/μL. Fever was defined as an axillary temperature ≥ 37.5 °C based on a single measurement. Serum was collected within the first 72 hours after the onset of FN in all episodes. During some episodes, serum was collected as a control at the onset of fever, or before chemotherapy when the subjects were afebrile, in accordance with the protocol. Serum sCD14-ST was measured by the two-step enzyme-linked immunosorbent assay using recombinant sCD14-ST as the standards (Yaegashi Y, et al. J Infect Chemother. 2005;11:234-8). Patients with FN were classified into the following four groups: fever of unknown origin, local infection, bacteremia without hypotension, or septic shock. Results A total of 43 patients were eligible and 75 febrile episodes were evaluable. The median age of the patients (44% were male) was 45 years (range: 16–65). Seventy-five blood samples were collected within 72 hours after onset of fever (31 from 0–24 hours, 34 from 24–48 hours, and 10 from 48–72 hours), 12 while afebrile before chemotherapy, and 25 at the onset of fever. The corresponding serum sCD14-ST measurements were termed sCD14-ST&lt;72hr, sCD14-STcon, and sCD14-STonset, respectively. The underlying diseases included acute leukemia (56%), myelodysplastic syndrome (9%), non-Hodgkin’s lymphoma (16%) and others (19%). The median neutrophil count at study enrollment was 67/μL (range 0 to 962). The types of treatment prior to study entry included 49 (65%) chemotherapies, one (1%) autologous hematopoietic cell transplant, and 18 (24%) allogeneic hematopoietic cell transplants. The mean sCD14-STonset value (790 pg/ml, range: 364–1582, n=25) was significantly higher than that for sCD14-STcon (436 pg/ml, range: 262–846, n=12) [unpaired t-test, P&lt;0.001]. In 12 patients with control data, sCD14-ST&lt;72hr was significantly higher than sCD14-STcon (paired t-test, P&lt;0.001). In 25 patients with onset data, sCD14-ST&lt;72hr was significantly higher than sCD14-STonset (paired t-test, P&lt;0.047). The median sCD14-ST&lt;72hrwith fever of unknown origin (n=31), local infection (n=27), bacteremia without hypotension (n=9), septic shock (n=5) and others (n=3) was 680 (314–1986) pg/ml, 763 (348–2615), 782 (482–1550), 1359 (493–2115), and 738 (387–1402), respectively. Conclusion Serum levels of sCD14-ST may be a sensitive indicator of the development of FN. Within 72 hours of the onset of FN, serum levels of sCD14-ST appeared to increase. In addition, sCD14-ST might be a useful marker in detecting potentially lethal septic shock. Further prospective research in a large cohort is warranted to determine not only the diagnostic performance of this test but also its clinical utility as a tool for guiding antibiotic use, including the administration, timing and completion of these drugs in FN. Disclosures: Nakamae: Mochida Pharmaceutical Co., LTD.: Research Funding.

A second-generation ELISA (STRATIFY JCV™ DxSelect™) for detection of JC virus antibodies in human serum and plasma to support progressive multifocal leukoencephalopathy risk stratification

BackgroundJC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). The development and validation of a two-step enzyme-linked immunosorbent assay (ELISA) that detects JCV antibodies in human serum or plasma and its clinical utility for stratification of PML risk have been described. ObjectiveTo develop a second-generation JCV antibody ELISA kit with improved assay performance characteristics. Study designThe assay design was optimized by pre-coating the JC virus-like particles (VLP) on microtiter plates. Assay cut-points were statistically established using sera from >1300 multiple sclerosis patients from natalizumab clinical studies. The assay was analytically validated and then used to determine the presence of JCV antibodies in both treatment-naïve and natalizumab-treated MS patients, as well as in natalizumab-treated PML patients. ResultsAn improved assay for detection of JCV antibodies in human serum and plasma was developed. Key enhancements included improved delineation and reproducibility of low JCV antibody responses and assay ease of use. The assay was validated, demonstrating good agreement with the original two-step JCV antibody ELISA, and similar seroprevalence of 50%–60%. Samples from 63 natalizumab-treated PML patients collected 6–180 months prior to PML diagnosis tested JCV antibody positive. One patient tested JCV antibody negative 15 months prior to PML diagnosis but JCV antibody positive 2 months prior to PML diagnosis. ConclusionsThe validated second-generation JCV antibody ELISA offers improved assay design as a kit and enhanced performance characteristics that advance routine clinical use of the assay as a PML risk stratification tool.

Best practice in the use of natalizumab in multiple sclerosis

Natalizumab is approved for the treatment of patients with relapsing-remitting multiple sclerosis who have failed first-line treatment with traditional disease-modifying therapies or who have highly active disease. The drug has proved highly effective, both in a clinical trial setting and in clinical practice, with marked reductions in the rate of clinical relapses and slowed disease progression. These clinical outcomes are mirrored by a marked reduction in disease activity as evidenced by magnetic resonance imaging of the brain. However, natalizumab treatment has been associated with a risk of progressive multifocal leukoencephalopathy (PML), a potentially fatal condition caused by JC virus (JCV) activation. When this condition was detected in a clinical trial shortly after approval, the drug was immediately and voluntarily withdrawn from the market. As a condition of its reinstatement, stringent pharmacovigilance measures and a risk management plan were enforced. The recent availability of a two-step enzyme-linked immunosorbent assay (ELISA) test for the presence of anti-JCV antibodies (free testing is available in a central laboratory for registered centers), along with an ever-improving understanding of other risk factors such as prior immunosuppressant use and duration of treatment, allow an increasingly refined stratification of the risk of PML. This improved stratification of risk can help guide decisions about treatment. This review will also deal with other topics of relevance to clinical practice such as the development of antinatalizumab antibodies and their negative implications in terms of hypersensitivity reactions and loss of efficacy, withdrawal of treatment, and compassionate pediatric use.

The effect of plasma exchange on serum anti-JC virus antibodies

Objective: Natalizumab, a highly effective treatment for multiple sclerosis (MS) and Crohn’s disease, is associated with progressive multifocal leukoencephalopathy (PML). Upon suspicion or diagnosis of PML, plasma exchange (PLEX) is performed to remove natalizumab from the circulation, allowing immune reconstitution of the central nervous system. Since PLEX may also remove other circulating antibodies, we examined the effects of PLEX on serum immunoglobulin (IgG) and anti–JC virus (JCV) antibody levels in MS patients with and without PML. Methods: Serum samples from 12 natalizumab-treated patients without PML collected before, during and after PLEX were tested for IgG isotypes using a commercial assay, and for anti-JCV antibodies using a two-step enzyme-linked immunosorbent assay. Five natalizumab-treated PML patients who underwent PLEX were also tested for anti-JCV antibodies. Results: PLEX produced a two- to three-fold reduction in all IgG isotypes. Among patients without PML, 42% (five of 12 patients) had detectable anti-JCV antibodies before PLEX; in these patients, anti-JCV antibodies were reduced approximately two- to five-fold, with levels returning to 50–100 percent of baseline two weeks after the final PLEX. The five PML patients, all of whom had detectable anti-JCV antibodies before PLEX, experienced similar reductions in anti-JCV antibody levels following PLEX. Conclusions: Our results indicate that PLEX effectively removes circulating antibodies; however, levels of endogenous anti-JCV antibody, unlike exogenously administered natalizumab, were replenished relatively quickly following PLEX. While interpretation of anti-JCV antibody levels during or within two weeks after PLEX may be problematic, humoral JCV immunity is not abolished by PLEX and antibody levels are rapidly restored.

Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma

BackgroundJC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification. ObjectivesTo validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method. Study designAnalytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability. ResultsAnalytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples. ConclusionsThe novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum

BackgroundWe compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad).MethodsWe tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included.ResultsThe sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%).ConclusionOur findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.

Sensitive enzyme-linked immunosorbent assay for adult T-cell leukemia-derived factor and normal value measurement.

Four different monoclonal antibodies against recombinant adult T-cell leukemia-derived factor (ADF), identical to thioredoxin, were established and used for the determination of ADF concentration in serum. Using two of the monoclonal antibodies, we developed a two-step enzyme-linked immunosorbent assay (ELISA) for ADF. This ELISA showed a highly specific reactivity on ADF with no cross-reactivity to several proteins with homologue sequence on the active center. The detection limit of the assay was 2.0 ng/ml (mean +/- 2 SD). The intra- and interassay coefficients of variation (CV) were 0.81-3.74% (n = 8) and 4.78-6.97% (n = 7), respectively. The normal value of ADF mean concentration from 145 healthy donors was 40.8 ng/ml.

Development of a class-specific competitive enzyme-linked immunosorbent assay for the detection of pyrrolizidine alkaloids in vitro

A two-step enzyme-linked immunosorbent assay (ELISA) was developed for the determination of the presence of retronecine in the parts per billion range. By modification of retronecine two haptens were synthesized. An 150 value of 11 f 3 ppb (Figure 2a) was obtained, with detection limits of 1.0-100 ppb. Indirect detection of monocrotaline (325 g/mol), retrorsine (351 g/mol), and senecionine (352 g/mol) was achieved by hydrolytic digestion of these naturally occurring pyrrolizidine alkaloids (PAS), demonstrating the utility of the assay as a class-specific detection method. PAS were detected in Senecio vulgaris and Crotalaria retusa plant sample extracts. No cross-reactivity was seen with either swainsonine or lupinine, both of which are structurally similar to retronecine in that they are also fused bicyclic alkaloids.

Quantitation of immunoglobulins in mouse tears using enzyme linked immunosorbent assay (ELISA).

Aqueous tears were collected by glass capillary tubes at weekly intervals from 6-9 week old, Swiss-Webster mice. Mouse IgA, IgG1, IgG2a and IgM were quantitated in the tears using sandwich type enzyme linked immunosorbent assays (ELISAs). IgE was quantitated using an indirect two-step ELISA. The results established IgA as the predominant antibody in mouse tears at a level of 110 micrograms/ml. This was determined using a mouse IgA standard which contained both polymeric and monomeric IgA. IgG1, IgG2a and IgM were detected in the tears at low levels, 13 ng/ml, 21 ng/ml and 442 ng/ml, respectively. IgE was not detectable in mouse tears with a lower limit of detection equal to 10 ng/ml. These studies establish the relative levels of IgA, IgG1, IgG2a and IgM in the tears of normal adult mice.