Evaluation of various radioimmunological techniques for the detection of antigen HBs]

The different radioimmunoassay technics routinely used for detection of HBs Antigen are Ausria I, Ausria II, RIA and CLB/RIA. Classification of these techniques either liquid or solide phase, is realized by nature of the tracer (Antigen or Antibody radioiodinated). The sensitivity and the specificity are presented as the comparison with other reactions. There is no significative difference on 1089 tested samples between Ausria I and RIA. The percentage of false positives in RIA or CLB/RIA reactions is 1.5% when the incubation is at 20 degrees C and 1% when it is at 37 degrees C. Advantages, disadvantages, cost prices, automation and possibility of computer treatment are discussed. The utilization of reference sera is proposed and the problem of the utilization en France of the radioactive products is approached.     

Application of a reversed passive hemagglutination test to the detection of HBs antigen]

In this report we present an evaluation of the sensitivity, specificity and ability to detect HBs Ag carriers of a new reversed passive hemagglutination test, using immunochemically purified chimpanzee anti HBs bound to stabilized human erythrocytes. The method was shown to have a sensitivity equal (within one two fold dilution) to that of the Ausria I 125 ratio immuno assay, and in a double blind comparison detected essentially the same number of Hbs Ag containing specimens among volunteer blood donors. The method therefore provides an economical method for the third generation testing of blood donors. The methodology which has been described incorporates a definitive specificity test in which serum drawn before and after immunization of chimpanzees with purified HBs Ag is compared for its ability to neutralize the hemagglutination reaction. The use of serum from the same animal for this purpose avoids the theoretical possibility that antiglobulin antibodies directed at subclass determinants such as Gm of Inv could be differentially inhibited due to possible subclass differences in the blocking sera employed. A reliable test for specificity of HBs Ag screening results is essential to avoid false notification of donors that they are carriers of hepatitis B virus.     

Comparative study of three commerical preparations for the detection of Australia antigen]

2,944 sera from blood donors (420 of them being new donors) were tested in four techniques: Counterelectrophoresis (CEP) and three commercial tests: Ausria II, Auscell and Hepanosticon. Over 10 HBs Ag detected by RIA, 8 were evidenced by the Auscell test and 6 by the Hepanosticon Test. False positive results (approximately 3%) represent a disadvantage to the reverse hemagglutination method; this rate increased considerably in a group of 386 patients sera also tested. The authors conclude that the results obtained with the reverse hemagglutination technique are nearing those of RIA.     

Plasma miRNAs as Diagnostic and Prognostic Biomarkers for Ovarian Cancer

Background

Most (70%) epithelial ovarian cancers (EOCs) are diagnosed late. Non-invasive biomarkers that facilitate disease detection and predict outcome are needed. The microRNAs (miRNAs) represent a new class of biomarkers. This study was to identify and validate plasma miRNAs as biomarkers in EOC.

Methodology/Principal Findings

We evaluated plasma samples of 360 EOC patients and 200 healthy controls from two institutions. All samples were grouped into screening, training and validation sets. We scanned the circulating plasma miRNAs by TaqMan low-density array in the screening set and identified/validated miRNA markers by real-time polymerase chain reaction assay in the training set. Receiver operating characteristic and logistic regression analyses established the diagnostic miRNA panel, which were confirmed in the validation sets. We found higher plasma miR-205 and lower let-7f expression in cases than in controls. MiR-205 and let-7f together provided high diagnostic accuracy for EOC, especially in patients with stage I disease. The combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection. MiR-483-5p expression was elevated in stages III and IV compared with in stages I and II, which was consistent with its expression pattern in tumor tissues. Furthermore, lower levels of let-7f were predictive of poor prognosis in EOC patients.

Conclusions/Significance

Our findings indicate that plasma miR-205 and let-7f are biomarkers for ovarian cancer detection that complement CA-125; let-7f may be predictive of ovarian cancer prognosis.     

A liquid-phase immunoradiometric assay (IRMA) for human sex hormone binding globulin (SHBG)

An immunoradiometric assay (IRMA) for sex hormone binding globulin (SHBG) has been developed in which an 125I-labeled monoclonal antibody [( 125I]S1B5) and a rabbit anti-SHBG antiserum (RAb) are incubated in "liquid-phase" with standards or samples, and RAb-bound complexes are separated using donkey anti-rabbit IgG antibody-coated cellulose. This immunoassay technique is characterized by several advantages; the [125I]S1B5 imparts additional specificity and obviates the requirement for pure SHBG; the use of excess reagents reduces incubation times and also improves assay performance and sensitivity, and incubation in "liquid-phase" conserves and increases the efficiency of the RAb. The assay measures only non-denatured SHBG and is not influenced by the presence of steroid at the binding site. Assay specificity was demonstrated by parallelism between dilutions of pure SHBG and different serum samples. The quantitative recovery of SHBG added to serum, and the agreement between specific activities of SHBG in pure standards and sera, confirm the accuracy of the method. The within and between assay coefficients of variation were less than 7% and less than 11%, respectively, between 12 and 450 nmol/l. The assay sensitivity may be manipulated by altering the concentration of RAb and/or by preincubation with either [125I]S1B5 or RAb, and 0.2 fmol SHBG may be measured on a standard curve. The SHBG assay has been used to measure SHBG concentrations in sera, amniotic fluid, cerebral spinal fluid, seminal plasma and saliva.     

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative,Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing^TM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.     

A radioreceptor assay for insulin: direct measurement of dog pancreatic vein serum insulin.

A simple radioreceptor assay for insulin rat liver membranes as receptor sites, with sufficient specificity precision, and sensitivity to detect 10 ng or 276 muU/ml of serum insulin, has been developed. In the presence of standard porcine insulin at the concentration of 1.0 ng/tube, approximately 8% of 125I-porcine insulin was bound to the plasma membranes and ninety-five per cent of this binding was inhibited by 1.0 microgram of standard insulin per tube. Four animal insulins inhibited the binding of 125I-insulin while ACTH, glucagon, human growth hormone, and oxytocin were inert. Insulin values in dog pancreatic vein sera obtained during and after glucose loading and measured by the present radioreceptor assay agreed well with immunoreactive insulin. The ratio of IRI to the measurement by radioreceptor assay was 1.09 +/- 0.18 for the same sera.     

A ribonuclease protection assay for the direct detection and quantitation of hepatitis C virus RNA

A ribonuclease protection assay (RPA) was developed for the direct detection and quantitation of HCV RNA in infected patients' sera or plasma using HCV [(32)P]RNA from the conserved 5'-untranslated region (5'-UTR) as a probe. We were able to directly detect the presence of HCV RNA by RPA in several infected patients' samples. The viremic status of HCV infected patients with indeterminate recombinant immunoblot assay (RIBA II) was also determined by this assay. Polymerase chain reaction (PCR) was also performed on all these samples and were found to be positive with a concordance of 100% between the results of PCR and RPA. The RPA was able to detect approximately 1 pg of HCV RNA. A limited sequence heterogeneity among HCV isolates was also observed by this assay, suggesting that this may be a faster method of detecting heterogeneous HCV sequences in patients' samples. This simple and specific method could be used to quantitate HCV RNA in order to better determine viremia and follow the course of HCV infection especially when RIBA II results are indeterminate.     

Inhibition of lymphokine-activated killer cell generation by blocking factors in sera of patients with head and neck cancer

Summary Cytolytic activation of peripheral blood lymphocytes by recombinant interleukin-2 (rIL-2) in patients with squamous cell carcinoma (SCC) of the head and neck may be inhibited by serum blocking factors, and this could influence therapeutic efficacy. Peripheral blood lymphocytes from 21 patients with this disease and 17 controls were incubated with 10–1000 U rIL-2 for 6 days in supplemented complete medium (containing 10% fetal calf serum) or the same medium plus 10% autologous serum. After washing the effector cells, we determined their cytotoxicity against K562 and MDA1386, a lymphokine-activated-killer(LAK)-sensitive SCC cell line, using a⁵¹Cr-release assay. Patient sera inhibited LAK-generated lysis of both MDA 1386 and K562, while control sera from healthy persons inhibited LAK-generated lysis of MDA1386. The blocking activity of patient sera tended to be greater than that of control sera. The sera of patients with untreated or recurrent disease and those who were free of disease had equivalent inhibitory capacity. The serum blocking factor acted in a dose-dependent manner, and inhibition was overcome by increasing the dose of rIL-2. Levels of circulating immune complexes (measured by the Clq binding method) did not correlate significantly with inhibition. A clinical protocol of repeated plasma exchange in patients with advanced and recurrent squamous cell carcinoma of the head and neck allowed sequential study of one patients's serum before, during, and after treatments. Plasmapheresis removed serum inhibitory factors, albeit temporarily. The activity of serum blocking factors in patients with this disease can be modulated by increasing doses of rIL-2 and by plasma exchange. This modulation may be important to improving clinical response rates for patients undergoing immunotherapy.     

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.     

The potential value of CA125 as a tumour marker in small volume, non-evaluable epithelial ovarian cancer.

Seventy four consecutive patients with epithelial ovarian cancer have been followed up longitudinally with serial serum CA125 for up to 48 months. From this database, the CA125 changes in small volume disease have been evaluated. For long term complete responders (n = 12), the mean plateau level of CA125 was 7.2 U/ml (95% confidence interval; 5.6 to 9.2 U/ml). The natural half-life of CA125 at 5.1 days (range 3.8 to 7 days) was calculated from five patients with Stage I and II disease who underwent complete surgical excision. A mean lead time of 99 days (range 14 to 255 days) was demonstrated between marker detection of disease progression and clinically apparent progressive disease in 12 out of 13 patients (92%) who relapsed after chemotherapy induced complete remission. The threshold of tumour volume detection with CA125 is unlikely to be determined by an arbitrary cut-off level. The kinetics of CA125 provide more useful information and the potential to define complete response or indeed cure with CA125 parameters requires further investigation.     

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 μl serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a ³²P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.     

High-performance liquid chromatographic determination of pyrazoloacridine, a nitro-9-methoxyacridine anticancer agent, in human plasma

Pyrazoloacridine (PZA) is a 9-methoxy substituted acridine with a reducible nitro group. PZA has shown selective solid tumor cytotoxicity with activity against hypoxic cells, non-cycling cells and cells expressing the multidrug resistant phenotype. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of PZA in human plasma to support phase II clinical trials. PZA and ethyl orange, the internal standard, were isolated from human plasma by precipitating plasma proteins with methanol, and centrifuging to pellet the proteins. The resulting supernatant was injected onto a cyanopropyl HPLC column eluted isocratically with a mobile phase consisting of 125 mM ammonium acetate buffer pH 4.75-acetonitrile (76:24, v/v). A single wavelength at 460 nm was used for detection. Relative standard deviations for the assay ranged from 5.0% to 12.2% for four different drug concentrations and the limit of quantitation was 100 ng/ml. During the validation short term stability of the drug in plasma and stability of PZA on repeated freezing and thawing of plasma was evaluated. Overall recovery of PZA was 88%. This simple assay was found suitable for studying the clinical pharmacokinetics of PZA.     

Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 125I-protein A from Staphylococcus aureus.

Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with 125I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, resulted of the Staph A assay correlated highly (r = 0.981, p less than 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of 125I-Staph A in the solid phase radioimmunoassay was superior to 125I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the 125I Staph A assay may be applied ot IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.     

Persistence of antibodies of the IgG class against cytomegaloviruses in blood donors

High titre plasmas gained from blood donors are the initial material used for producing human immunoglobulin containing cytomegalovirus antibodies (CMV-HIG). For this purpose the sera gained from 467 permanent donors at the District Institute for Blood and Transfusion Service in Berlin were investigated on their CMV antibody content of IgG class by means of an indirect immunofluorescence test (IFT). The infection rate of blood donors amounted to 62% (291/467). CMV-IgG titre greater than or equal to 1:40 was determined in 88 sera (18.8%) and greater than or equal to 1:160 in 14 sera (3%). Two CMV-HIG laboratory samples (charges 3113 and 3117) were produced from these plasmas. Particularly immunoglobulin fraction of charge 3117 (No. 3117 N II) revealed excellent antibody titres (IFT 1:640 [CMV-IgG] or 1:40 [CMV-IgM] respectively, neutralisation test 1:32). Checks made with the donors' CMV-IgG titres after repeated application of plasmapheresis resulted in maximal titres changes of two dilution stages in a period of 15 months. Thus, in producing CMV-HIG a sure, tested pool of donors can be resorted to.     

The production of a “universal developer” for the immunological detection of human IgG and its application in immunodiagnostics

Summary This study describes the development of a bispecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fe portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG₁ subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbant assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.This study demonstrates the potential use of bi-specific universal developers in a wide variety of immunobased techniques as well as the potential advantages for the production of a complete panel of bi-specific developing monoclonal antibodies against IgGs from a number of different species.     

A modification in technology improves the specificity of the Du Pont de Nemours immunoenzyme kit for the detection of anti-HIV antibodies

Du Pont De Nemours perfected a new enzyme immunoassay for the screening of anti-HIV 1 antibodies, characterized by its rapidity: 2 hours of incubation, when the normal procedure needed 3 hours 30 minutes of incubation. We tested with the normal procedure and the shortened one 4,025 randomly selected blood donors. 55 samples of the SNTS panel and 10 sera representing the Western-Blot reference panel of the "Retrovirus" study group of the SNTS. We can say that shortening the incubation time results in certain advantages opposite the previous procedure: time saving (1 hour 30 minutes); improved specificity: the rate of false positive, 0.37% with the normal method, drops to 0.07% with the short one; good sensitivity, since all the individual positive samples were detected but it seems slightly less sensitive than the normal method for the diluted positive samples.     

Rapid determination of tafenoquine in small volume human plasma samples by high-performance liquid chromatography-tandem mass spectrometry

A method was developed for the determination of tafenoquine (I) in human plasma using high-performance liquid chromatography-tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with methanol containing [2H3(15N)]tafenoquine (II) to act as an internal standard. The supernatant was injected onto a Genesis-C18 column without any further clean-up. The mass spectrometer was operated in the positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring mode. The assay required 50 microl of plasma and was precise and accurate within the range 2 to 500 ng/ml. The average within-run and between-run relative standard deviations were < 7% at 2 ng/ml and greater concentrations. The average accuracy of validation standards was generally within +/- 4% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze-thaw cycles and samples can safely be stored for at least 8 months at approximately -70 degrees C. The method was very robust and has been successfully applied to the analysis of clinical samples from patients and healthy volunteers dosed with I.     

A 125I-protein A-binding assay detecting antibodies to cell surface antigens

A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were then investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells.     

Cell-mediated immunity to Friend virus-induced leukemia. I. Modification of 125IUdR release cytotoxicity assay for use with suspension target cells.

Cell-mediated cytotoxic reactivity of C57BL/6 mice against syngeneic FBL-3 cells, a Friend virus-induced leukemia, was measured by the 125IUdR release assay with tumor target cells in suspension. The tests could be performed either with Linbro tissue culture plates(16-mm wells) or with Microtest II plates (6-mm wells). The former could only be harvested manually; the latter could be harvested mechanically by an automatic harvesting apparatus which permitted larger scale tests. With either plate, it was found that careful preparation of the target cells and of the attacker cells has important effects on the results obtained. When performed under optimal test conditions, the 125IUdR assay can be used as a very simple, objective, and reproducible assay for testing cell-mediated cytotoxicity.