Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.     

A simplified method of rapid hemagglutination test for determining the levels of tetanus antibodies]

A new, simplified methodology of preparation of lyophilized chrome-coated turkey blood cells (formalin-treated ) for rapid identification of tetanus antibodies in indirect hemagglutination test is described. Blood cells diagnostic preparations obtained in this way were easier and three times faster to prepare than tanned cell preparations . They maintained unchanged capability for specific agglutination during at least one year of storage when kept at 4 degrees C. proposed methodology enable to start a production of laboratory kits necessary for controlled prophylaxis of tetanus.     

Study on adsorption of Cu(II) by water-insoluble starch phosphate carbamate

Crosslinked starch phosphate carbamates were prepared and used to adsorb Cu(II) ions from an aqueous solution. Scanning electron microscopy (SEM) was used to investigate the changes in the starch granule structure before and after adsorption. Batch adsorption experiments were carried out as a function of adsorption time, adsorbents dose, pH, substitute groups' content, initial Cu(II) ions concentrations, and temperature. The results reveal that 20 min of adsorption time is sufficient for reaching the adsorption equilibrium, the adsorption of Cu(II) ions on crosslinked starch phosphate carbamate is endothermic in nature, and the adsorption equilibrium data correlate well with the Langmuir isotherm model with the maximum adsorption capacity of 1.60 mmol/g. Moreover, the adsorbed Cu(II) ions can be desorbed by treating with HCl solution and the desorption percentage reached above 96% when desorbing with 1 N HCl solution for 1 h.     

Comparison of the indirect immunofluorescent (IFAT), ELISA test and the comercial Chagatek test for anti-Trypanosoma cruzi antibodies detection

Chagas disease is a public health problem in Colombia, particularly in the eastern region. Because of human migration from rural areas to urban centers, the possibility of transfusional transmission becomes increasingly important. However the risk can be minimized by a careful screening of blood donors by means of serological tests. Colombian blood banks use comercial, foreign serological tests for screening for T. cruzi infection. The purpose of the current study was to compare the IFAT and ELISA tests (both use antigen obtained from Colombian strains) with the comercially available Chagatek tests. Sera of blood donors were classified in two groups on the basis of the IFAT: group I, 15 positive patients and group II, 14 negative patients. Sera from each group were tested by the ELISA and Chagatek tests. The ELISA test detected 100% of the patients as positive in group I and 7% (1/14) of patients as positive in group II. The Chagatek test detected 93% (14/15) of the patients as positive in group I and 50% (7/14) in group II. The kappa index for concordance between the ELISA and IFAT tests was 0.93 (95% C.I.: 0.80-1.00); between IFAT and Chagatek 0.43 (95% C.I.: 0.26-0.62), and between ELISA and Chagatek 0.49 (95% C.I.: 0.31-0.67). These results highlighted the importance of using autochtonous Colombian strains as antigens in screening tests for blood donors.     

Evaluation of an indirect fluorescent antibody test (IFAT) for demonstration of antibodies to Toxoplasma gondii in the sea otter (Enhydra lutris)

An indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii infection was validated using serum from 77 necropsied southern sea otters (Enhydra lutris nereis) whose T. gondii infection status was determined through immunohistochemistry and parasite isolation in cell culture. Twenty-eight otters (36%) were positive for T. gondii by immunohistochemistry or parasite isolation or both, whereas 49 (64%) were negative by both tests. At a cutoff of 1:320, combined values for IFAT sensitivity and specificity were maximized at 96.4 and 67.3%, respectively. The area under the receiver-operating characteristic curve for the IFAT was 0.84. A titer of 1:320 was used as cutoff when screening serum collected from live-sampled sea otters from California (n = 80), Washington (n = 21), and Alaska (n = 65) for T. gondii infection. Thirty-six percent (29 out of 80) of California sea otters (E. lutris nereis) and 38% (8 out of 21) of Washington sea otters (E. lutris kenyoni) were seropositive for T. gondii, compared with 0% (0 out of 65) of Alaskan sea otters (E. lutris kenyoni).     

The effects of placing small amounts of phosphate fertilizer close to the seed on growth and nutrient concentrations of lettuce

Summary Two glasshouse experiments are described in which the effects of applying starter phosphate fertilizer, 1 cm beneath the seeds, on early growth and nutrient concentrations of lettuce (Lactuca sativa L. cv. Avondefiance) in well fertilized soil were determined. In Experiment 1 various rates of starter P in the form of NH₄H₂PO₄ were applied to soil containing a range of rates of incorporated triple superphosphate. Although there was little response of lettuce dry weight to the incorporated triple superphosphate there was a large response (about 65% increase after 36 days) to the starter. N and P concentrations within the plants were increased by the starter treatments whereas K concentration was reduced. The per cent P in the plants at 36 days from sowing could account for 60% of the variation in plant dry weight. In Experiment 2 the starter P was added as either the Ca, Na or K salt, with or without added (NH₄)₂SO₄. Adding the starter P without ammonum increased the P concentration of the plants by an average of 12% and the dry weight by an average of 39% at 30 days from sowing. The addition of ammonium ions increased plant concentrations of P, Mg and N but decreased plant K concentration. The effect of the ammonium ions on growth depended on the form of phosphate supplied as the starter. This variation in effect of ammonium ions was attributed to the effects of other starter ions on the relative concentration of ammonium in the soil solution.     

Grid-immunoblotting: a fast and simple technique to test multiple allergens with small amounts of antibody for cross-reactivity

Grid-immunoblotting is a procedure that allows the simultaneous testing of up to 20 different antibodies such as monoclonal antibody-containing hybridoma supernatants or human sera for specific antibodies to up to 20 different antigens or allergens on a single sheet of nitrocellulose membrane. Since only 150 to 200 μl of antibody-containing solution are required this technique is uniquely suited to test growing hybridomas and small amounts of sera (e.g. mouse and children’s sera). Compared to a standard ELISA, approximately ten times less antibody is needed to obtain the same information.     

Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39

Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H(2)O(2)) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H(2)O(2) production. Unexpectedly, ΔackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H(2)O(2) and AcP levels, and secondary capsule mutations in cps2E or cps2C. ΔackA ΔspxB mutants contained half the cellular amount of ATP as a ΔspxB or spxB(+) strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ΔackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H(2)O(2) may require energy. Starting with a ΔspxB mutant, we constructed Δpta, ΔackA, and Δpta ΔackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK(Spn), CiaRH(Spn), and LiaSR(Spn) TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.     

Hydrolysis of phosphatidylcholine by cerium(IV) releases significant amounts of choline and inorganic phosphate at lysosomal pH

Niemann-Pick disease and drug-induced phospholipidosis are examples of lysosomal storage disorders in which serious respiratory infections are brought on by high levels of the phospholipid phosphatidylcholine in the acidic lamellar bodies and lysosomes of pulmonary cells. One approach to developing an effective therapeutic agent could involve the use of a metal to preferentially hydrolyze phospholipid phosphate ester bonds at mildly acidic, lysosomal pH values (~ pH 4.8). Towards this end, here we have investigated phosphatidylcholine hydrolysis by twelve metal ion salts at 60 °C. Using a malachite green/molybdate-based colorimetric assay to detect inorganic phosphate released upon metal-assisted phosphate ester bond hydrolysis, Ce(IV) was shown to possess outstanding reactivity in comparison to the eleven other metals. We then utilized cerium(IV) to hydrolyze phosphatidylcholine at normal, core body temperature (37 °C). The malachite green/molybdate assay was used to quantitate free phosphate and an Amplex® Red-based colorimetric assay and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were employed to detect choline. Ce(IV) hydrolyzed phosphatidylcholine more efficiently at lysosomal pH: i.e., at a Triton X-100:phosphatidylcholine molar mixing ratio of 1.57, yields of choline and phosphate were 51 ± 4% and 40 ± 4% at ~ pH 4.8, compared to 28 ± 4% and 27 ± 5% at ~ pH 7.2.     

Clinical significance of platelet alloantibodies detected in immunofluorescence test (neonatal alloimmune thrombocytopenia and post-transfusion purpura)

Three cases of neonatal alloimmune thrombocytopenia and one patient with post-transfusion purpura could be diagnosed only by introducing the platelet immunofluorescence test. Thrombocytopenia was caused by anti-PlA1 platelet alloantibodies detected neither in the agglutination nor by the complement fixation test.     

Phospholipase A(2) activity towards vesicles of DPPC and DMPC-DSPC containing small amounts of SMPC.

Phospholipase A(2) (PLA(2)) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA(2) towards large unilamellar vesicles composed of DPPC:SMPC and DMPC:DSPC:SMPC is investigated using fluorescence and HPLC techniques. Phase diagrams of the mixtures are established by differential scanning calorimetry and the PLA(2) activity, monitored by the lag time, is correlated with the phase behavior of the mixtures. In addition, the degree of lipid hydrolysis in the DMPC:DSPC:SMPC lipid mixtures is detected by HPLC. The PLA(2) activity is found to be significantly increased in the temperature range of the coexistence region where the lipid mixtures exhibit lateral gel-fluid phase separation. Furthermore, in the entire temperature range it is demonstrated that PLA(2) preferentially hydrolyzes the short chain DMPC lipid. This discriminative effect becomes less pronounced when the asymmetric lipid SMPC is present in the lipid substrate. Inclusion of SMPC into either DPPC or DMPC:DSPC vesicles prolongs the lag time. The results clearly show that the PLA(2) activity is significantly enhanced by lipid bilayer micro-heterogeneity in both one-component and multi-component lipid bilayer substrates. The PLA(2) activity measurements are discussed in terms of dynamic gel-fluid lipid domain formation due to density fluctuations and static lipid domain formation due to gel-fluid phase separation.     

Extraction and labeling methods for microarrays using small amounts of plant tissue

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5–30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng–7 μg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).