Simple determination of polysaccharide specific antibodies by means of chemically modified ELISA plates.

A new ELISA technique using Nunc CovaLink NH microtiter plates has been developed to measure anticapsular polysaccharide specific antibodies. Capsular polysaccharide (PS) of Haemophilus influenzae type b (PRP) and pneumococcal antigens types 3, 6, 8, 14, 19, 23 were immobilized on CovaLink NH. These are modified plates with secondary amino groups bound to their surface which, in the presence of a water-soluble carbodiimide as coupling reagent, facilitate the direct binding of polysaccharides. We compared the binding characteristics of PS antigens to CovaLink NH and a conventional polystyrene ELISA plate. Checkerboard titration of PS antigens between 0.04-30 micrograms/ml clearly demonstrated that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 microgram/ml, while binding of PS to the conventional plate was rather poor even at concentrations of 30 micrograms/ml. The CVs for the ELISA ranged from 1.1 to 2.8% for intra-assay comparisons and from 3.6 to 7.3% for inter-assay comparisons. In addition, when PRP-IgG antibodies were determined with the CovaLink NH ELISA and compared with the Farr assay an acceptable correlation ( r = 0.89, p < 0.0001) was obtained. The technique described provides a simple and sensitive tool for evaluating specific immunity to PS antigens.

Landomycins, new angucycline antibiotics from Streptomyces sp. I. Structural studies on landomycins A-D.

The chemical structure of the new angucycline antibiotic landomycin A has been elucidated via chemical and spectroscopic methods, in particular by 2D NMR correlation spectroscopy, e.g., 1H, 1H-COSY, 13C, 1H-COSY, correlation spectroscopy via long-range-couplings and heteronuclear multiple bond connectivity spectroscopy sequences. The spectroscopic investigations were carried out principally with the octaacetyl derivative of landomycin A, which is more soluble in organic solvents than landomycin A itself. The structure consists of a new, unusual angucyclinone, landomycinone A, and of six deoxy sugars, four D-olivoses and two L-rhodinoses, which are all assembled in one chain thus forming the sequence (olivose-4----1-olivose-3----1-rhodinose)2. This long sugar chain is bonded as a phenolic glycoside to the aglycone moiety, a unique structural feature among quinone glycoside antibiotics. By comparison with the main component landomycin A, the structures of three minor congeners, namely landomycins B, C and D, could be proposed.

Identification of Gibberellins in Norway Spruce (Picea abies [L.] Karst.) by Combined Gas Chromatography-Mass Spectrometry.

Gibberellins A(1) (GA(1)), A(3) and A(9) were identified from extracts of shoots of 6-month old Norway spruce (Picea abies) seedlings by the use of sequential reverse and normal phase high performance liquid chromatography (HPLC), bioassay, radioimmunoassay (RIA) and combined gas chromatography-mass spectrometry (GC-MS). The bioassay and RIA were used after fractionation by HPLC to detect the GA-containing fractions, which were then examined by GC-MS. The GAs identified are considered to be endogenous.

Comparison of gas chromatography-mass spectrometry, radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.).

Quantitative estimates of gibberellin A9 in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A9, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.

Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod.

Cell-free extracts capable of converting [(14)C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [(14)C]-labeled GAs, prepared enzymically from [(14)C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA(12) --> GA(53) --> GA(44) --> GA(19) --> GA(20). The pH optima for the enzymic conversions of [(14)C]GA(53), [(14)C]GA(44) and [(14)C]GA(19) were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe(2+), alpha-ketoglutarate and O(2) for activity, and ascorbate stimulated the conversion of [(14)C]GA(53) and [(14)C]GA(19). Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA(53) and GA(19) are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA(44) remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA(20) content of spinach plants in LD than in SD.

Conversion of [(14)C]gibberellin A 12-aldehyde to C 19- and C 20-gibberellins in a cell-free system from immature seed of Phaseolus coccineus L.

A cell-free system prepared from developing seed of runner bean (Phaseolus coccineus L.) converted [(14)C]gibberellin A12-aldehyde to several products. Thirteen of these were identified by capillary gas chromatography-mass spectrometry as gibberellin A1 (GA1), GA4, GA5, GA6, GA15, GA17, GA19, GA20, GA24, GA37, GA38, GA44 and GA53-aldehyde, all giving mass spectra with (14)C-isotope peaks. GA8 and GA28 were also identified but contained no (14)C. All the [(14)C]GA12-aldehyde metabolites, except GA15, GA24 and GA53-aldehyde, are known endogenous GAs of P. coccineus.