A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase

Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for high-throughput applications.     

Renin substrate in human amniotic fluid

Purified human amniotic fluid renin substrate (RS) was compared to purified plasma RS. RS in plasma and amniotic fluid were similar in molecular weight, isoelectric point and immunological properties.

Immunoreactivity of radio-iodinated amniotic fluid RS was lower than that of plasma RS. Measured by direct radioimmunoassay, RS-levels were only 10–22% of those obtained with indirect assay in 22 amniotic fluid samples. This difference suggests that amniotic fluid RS is less immunoreactive than plasma RS, possibly due to biochemical alteration or complex formation. No such difference in immunoreactivity was noticed in RS of decidual and placental cytosolic fraction.     

Dose dependence of mass and microcalcification detection in digital mammography: free response human observer studies

The purpose of this study was to evaluate the effect of dose reduction in digital mammography on the detection of two lesion types-malignant masses and clusters of microcalcifications. Two free-response observer studies were performed-one for each lesion type. Ninety screening images were retrospectively selected; each image was originally acquired under automatic exposure conditions, corresponding to an average glandular dose of 1.3 mGy for a standard breast (50 mm compressed breast thickness with 50% glandularity). For each study, one to three simulated lesions were added to each of 40 images (abnormals) while 50 were kept without lesions (normals). Two levels of simulated system noise were added to the images yielding two new image sets, corresponding to simulated dose levels of 50% and 30% of the original images (100%). The manufacturer's standard display processing was subsequently applied to all images. Four radiologists experienced in mammography evaluated the images by searching for lesions and marking and assigning confidence levels to suspicious regions. The search data were analyzed using jackknife free-response (JA-FROC) methodology. For the detection of masses, the mean figure-of-merit (FOM) averaged over all readers was 0.74, 0.71, and 0.68 corresponding to dose levels of 100%, 50%, and 30%, respectively. These values were not statistically different from each other (F= 1.67, p=0.19) but showed a decreasing trend. In contrast, in the microcalcification study the mean FOM was 0.93, 0.67, and 0.38 for the same dose levels and these values were all significantly different from each other (F = 109.84, p < 0.0001). The results indicate that lowering the present dose level by a factor of two compromised the detection of microcalcifications but had a weaker effect on mass detection.     

Prevalence of Human Papillomavirus in endometrial cancer: A systematic review and meta-analysis

Objective

HPV is a common sexually transmitted infection and is considered to be a necessary cause of cervical cancer. The anatomical proximity to the cervix has led researchers to investigate whether Human Papillomavirus (HPV) has a role in the etiology of endometrial cancer.

Methods

We conducted a systematic review and meta-analysis to investigate the pooled prevalence of HPV DNA in endometrial cancer. Using meta-regression, we further analyzed whether factors such as geographical region, HPV DNA detection method, publication year and tissue type were associated with HPV prevalence. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for studies providing data on HPV prevalence in cases with endometrial cancer and in controls with normal or hyperplastic endometrial tissue.

Results

We identified 28 papers (29 studies) examining the prevalence of HPV DNA in tumor tissue from endometrial cancer comprising altogether 1026 cases of endometrial cancer. The HPV prevalence varied considerably from 0% to 61.1%. From the random effects meta-analysis, the pooled prevalence of HPV DNA in endometrial cancer was 10.0% (95% CI: 5.2–16.2) with large between-study heterogeneity (I² = 88.2%, p < 0.0001). The meta-regression showed that HPV DNA detection method was statistically significantly associated with HPV prevalence (p = 0.0016): the pooled HPV prevalence was 6.0% (95% CI: 1.5–13.0) using general primers, 18.9% (95% CI: 8.6–32.1) using type-specific primers and 1.0% (95% CI: 0.0–3.6) using non-PCR based methods. None of the other a priori defined variables were statistically significantly associated with HPV prevalence. The pooled OR was 1.43 (95% CI: 0.68–3.00) indicating that the odds of HPV was not increased in cases versus controls.

Conclusions

HPV appears to have a limited or no role in the etiology of endometrial cancer.     

Evaluation of three commercial enzyme-linked immunosorbent assays and two latex agglutination assays for diagnosis of primary Epstein-Barr virus infection.

Three commercially available enzyme-linked immunosorbent assays (ELISAs) from Gull, Biotest, and Behring (Enzygnost) and two latex agglutination tests for heterophile antibodies (Monolatex [Biotest] and Mono-Lex [Trinity Laboratories]) were evaluated for the diagnosis of primary Epstein-Barr virus (EBV) infection and EBV seropositivity. Two hundred fourteen consecutive samples from 197 patients with symptoms of primary EBV infection were analyzed by the five assays at a clinical microbiology laboratory. The samples were also analyzed independently by immunofluorescence methods at a reference laboratory. According to the reference methods, 37 patients (40 serum samples) had primary EBV infections, 120 patients (127 serum samples) had had past EBV infections, 33 patients (36 serum samples) were seronegative, and 7 patients (11 serum samples) exhibited atypical reactions. The respective sensitivities and specificities for the diagnosis of primary EBV infection were 95 and 100% for the Gull assays, 100 and 94% for the Biotest assays, and 100 and 89%, for the Enzygnost assays. The Monolatex and Mono-Lex methods showed similar sensitivities and specificities (78 to 85% and 100 to 99%, respectively) for the diagnosis of primary EBV infection. This study demonstrates the usefulness of commercially available assays for the rapid diagnosis of primary EBV infection, but also the importance of large-scale testing of routine samples before choosing an assay.     

Cyclosporin A response and dependence in children with acquired aplastic anaemia: a multicentre retrospective study with long-term observation follow-up

Immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporin A (CyA) is the standard treatment for children with acquired aplastic anaemia (AAA) lacking a matched donor. Survival rates of more than 80% at 5 years are achieved, but the response is drug-dependent in 15–25% of cases. This study, of 42 consecutive children with AAA treated with IST, assessed the incidence of CyA-dependence, CyA and granulocyte colony-stimulating factor (G-CSF) tapering schedules and the impact of drug accumulation on progression to myelodysplasia/acute myeloid leukaemia (MDS/AML). Overall survival was 83% at 10 years. CyA-dependence without a predictive marker was observed in 18% of responders. Probability of discontinuing CyA was 60·5% at 10 years; a slow CyA tapering schedule was performed in 84% of patients; the cumulative incidence of relapse was 16% at 10 years. Relapse risk was significantly associated with rapid CyA discontinuation: 60% compared to 7·6% in the slow tapering group ( P  = 0·001). Cumulative incidence of MDS/AML was 8% at 10 years, with a significant correlation with both G-CSF cumulative dose and second IST. This long-term follow-up of children with AAA shows that IST with a slow CyA tapering course is an effective treatment with a low-relapse rate in these cases.