Antigenicity of topochemically related peptides.

Antibodies raised in rabbits against multimeric all-L peptides (MAP's) were first made monospecific by affinity chromatography on immobilized antigen columns and then tested for their ability to cross-react with topologically related variants of the parent antigen, where the chirality of each amino-acid residue (inverso derivatives), or the peptide sequence orientation (retro derivatives), was inverted, or where both modifications were simultaneously introduced (retro-inverso derivatives). Retro, inverso, and retro-inverso forms of the parent peptide were prepared, both in the linear as well as in the BSA-conjugated form, and found to cross-react to a significant extent with affinity purified polyclonal antibodies raised against the parent peptide. Peptide variants displayed similar dose-dependent inhibitory effects on the interaction between immobilized parent antigen and affinity purified antibodies. Analysis of molecular models of the peptide variants in the trans-configuration suggested that the topological equivalence of alternating side chains in the series of related peptides may be responsible for the observed cross-recognition, leading to the formation of similar recognition surfaces which could mimic the parent peptide antigenic structure.

Evidences that syngeneic alpha-type anti-idiotypic antibodies may non-competitively inhibit idiotype/oligomeric antigen interactions by affecting idiotype avidity.

The effects of syngeneic anti-Id antibodies on the multivalent interaction between human TNF-alpha, a homotrimeric Ag, and an anti-TNF mAb (mAb(1)78) have been studied. Eight anti-mAb(1)78 Ig secreting hybridoma, able to inhibit TNF binding in a competitive or non-competitive mode, have been generated. Two representative clones (mAb(2)1G3 and mAb(2)9F1) were selected for studying the inhibition mechanism of TNF-mAb(1)78 interaction. Idiotype-paratope topography studies indicated that mAb(2)1G3 (IgG2a) and mAb(2)9F1 (IgG1) bind two sterically distinct idiotopes on mAb(1)78 (IgG1) V regions. In particular, mAb(2)1G3 was found to bind an idiotope located within (or spatially close to) the Ag combining site suggesting that competitive inhibition of TNF binding to mAb(1)78 by mAb(2)1G3 occurs through paratope blockade. On the other hand, mAb(2)9F1 recognizes an idiotope located outside the paratope, being able to bind mAb(1)78 even in the presence of saturating amounts of TNF. mAb(1)78-TNF molar ratio in complexes, at stoichiometric equivalence, was unchanged in the presence of a large excess of mAb(2)9F1, suggesting that the functional bivalency of mAb(1)78 was not impaired by this anti-Id antibody. However, bivalent mAb(2)9F1 was able to partially inhibit the binding of bivalent mAb(1)78 to oligomeric TNF in liquid-phase as well as in solid-phase assays, whereas no inhibition was observed with monovalent mAb(2)9F1-F(ab) or mAb(1)78-F(ab). This suggests that inhibition is based on a decrease of the avidity of bivalent mAb(1)78 and not on allosteric effects on antigen binding sites. The effect of mAb(2)9F1 on mAb(1)78 arm flexibility and paratope orientation is discussed. In conclusion, the results indicate that anti-Id antibodies may inhibit Ag-antibody multivalent interactions by paratope blockade or by affecting the antibody avidity.

Antigenic regions of tumor necrosis factor alpha and their topographic relationships with structural/functional domains.

The immunogenic regions of human Tumor Necrosis Factor alpha (huTNF) have been mapped by studying the interaction between various mouse anti-huTNF sera and synthetic huTNF fragments, spanning the entire sequence of huTNF. Three main immunogenic regions were identified within residues 1-23, 95-116 and 137-157 of huTNF and two other less immunogenic regions within residues 117-136 and 37-55. The same huTNF regions were found to contain antigenic sites by binding studies with cognate anti-peptide sera. Competitive binding experiments with shorter synthetic subfragments provided evidence for the location of strong antigenic sites within residues 1-10, 17-23, 104-112 and 137-143. In particular the immunodominant site was found to be located within residues 104-112. huTNF regions corresponding to residues 24-36, 56-75, 76-94, and 147-157 resulted to be not or poorly antigenic. However, treatment of huTNF with Triton X-100 under conditions that partially dissociate the oligomeric quaternary structure resulted in the exposition of sites recognized by sera against peptides huTNF [56-75] and huTNF [76-94], suggesting that antigenic sites not accessible in the oligomeric huTNF are exposed in the dissociated form. The principal antigenic sites in the oligomeric molecule are localized in the flexible N-terminal part and in hydrophilic segments located in the "middle/top" region of the molecule, opposite to the C-terminus. Protein segments of the "bottom" region, close to the C-terminus, were poorly immunoreactive. Neutralization assays of TNF cytolytic activity on L-M cells showed that binding of antibodies to epitopes located in the "middle/top" regions of huTNF does not affect its cytolytic activity, supporting the hypothesis of a receptor binding site location at the "bottom" of TNF trimer.

Identification of two forms (31-33 and 48 kD) of the urinary soluble p55 tumor necrosis factor receptor that are differentially N- and O-glycosylated.

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

A rapid method for monitoring DNA labelling reactions with haptens.

A rapid and simple method for evaluating the efficiency of DNA labelling reactions with haptens is described. The method, called the Flow-Through Hapten-DNA Assay (FT-HDA), relies on binding of anti-hapten antibodies/alkaline phosphatase conjugates to hapten-DNA, immobilized on disposable capillary absorbent filters, and visual detection of blue-grey coloured spots appearing on the filter after chromogenic reaction with enzyme substrates. FT-HDA of hapten-DNA is markedly faster and simpler than conventional diffusion assays on membranes.

Tumor necrosis factor (TNF) alpha quantification by ELISA and bioassay: effects of TNF alpha-soluble TNF receptor (p55) complex dissociation during assay incubations.

It has been previously reported that different quantitative results can be obtained when TNF alpha is measured in biological fluids by bioassay and immunoassay. This is thought to be related to the presence of antigenic forms of TNF alpha that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNF alpha monomers. In this work we have observed discrepancies between antigenic and bioactive TNF alpha even when we used a sandwich-ELISA, unable to detect TNF alpha monomers, based on antibodies that bind epitopes overlapping with the soluble-receptor binding site of TNF alpha. Moreover, we found that antigenic TNF alpha levels in the presence of p55-sTNF-R (sTNF-R1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNF alpha-soluble receptor complex dissociation occurring during assay incubations contributes to the variability of results, we studied the kinetics of TNF alpha-soluble receptor interactions and examined the effect of complex dissociation using different analytical systems. TNF alpha association (k(on)) and dissociation (koff) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 x 10(5) s-1 M-1 and 2.8 x 10(-4) s-1, corresponding to an equilibrium constant (Kd) of 0.59 nM and to a half life for these complexes of 38 min. Complex dissociation and differential changes in the TNF alpha-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNF alpha quantification.

Differential detection of single-chain and two-chain urokinase-type plasminogen activator by a new immunoadsorbent-amidolytic assay (IAA).

A new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.

A monoclonal antibody that recognizes the receptor binding region of human urokinase plasminogen activator.

An anti-urokinase monoclonal antibody 5B4 (MAB 5B4) was obtained by fusing the murine myeloma cell line X63-Ag8.653 with the spleen cells from a female BALB/c mouse immunized with high-molecular-weight urokinase (HMW-uPA). MAB 5B4 is an IgG1 that binds selectively to the single-chain form of uPA (sc-uPA), to HMW-uPA and to the 17,000 Mr aminoterminal fragment of the A-chain (ATF) but not to the low-molecular-weight urokinase (LMW-uPA) nor to the reduced form of HMW-uPA. This strongly suggests that MAB 5B4 recognizes a conformational determinant on the A-chain. The antibody has an affinity constant for uPA-Sepharose of 1.42 X 10(7) M-1, calculated from equilibrium binding data, and can be used for one step purification of HMW-uPA by immunoaffinity chromatography. MAB 5B4 and the previously obtained antibody 105IF10 recognize the A-chain: the epitopes, however, are distinct as shown by double-antibody-sandwich enzyme immunoassay. Finally MAB 5B4 inhibits the binding of ATF to the uPA receptor of different human cells, whereas 105IF10 does not. Thus this antibody represents a potentially, useful tool for the study of uPA receptor physiology.

Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells.

A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.

The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1.

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase.

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Comparison of the solid phase enzyme receptor assay (SPERA) and the microbiological assay for teicoplanin.

A solid phase enzyme receptor assay (SPERA) for teicoplanin has been developed and recently presented in a kit form. The assay relies on the competition between antibiotic present in the biological fluid and peroxidase labelled teicoplanin for albumin-epsilon-amino-caproyl-D-alanyl-D-alanine (BSA-epsilon-ACA-D-Ala-D-Ala), a synthetic analog of the biological receptor. The kit contains BSA-epsilon-ACA-D-Ala-D-Ala coated microplates together with all the necessary reagents. The limit of detection of the SPERA is 0.5 mg 1(-1). The microbiological assay for teicoplanin uses Bacillus subtilis ATCC 6633 as test organism and a high salt medium to ensure a low limit of detection of 0.05 mg 1(-1). The previously established correlation between the two methods has been confirmed using the kits on 280 serum and 216 urine samples with concentrations up to 500 mg 1(-1).

Identification of differentially glycosylated forms of the soluble p75 tumor necrosis factor (TNF) receptor in human urine.

Human urine is known to contain a 30 kDa soluble form of the p75-TNF receptor (sTNF-R2). In this work we have purified sTNF-R2 from the urine of normal subjects and further characterized its structure and activity. sTNF-R2 was resolved by reducing SDS-PAGE in a major band of 30 kDa, similar in size to the previously described urinary sTNFR2, and in a minor band of 45 kDa. "Western" blotting analysis with anti-TNF-R1 and anti-TNF-R2 antibodies showed that both bands were immunologically related to the membrane TNF-R2. Glycosylation studies indicated that the 30 kDa is N-glycosylated while the 45 kDa form is N- and O-glycosylated, and suggested that both forms contain terminally linked sialic acid that is differentially recognized by lectins. These results indicate that human urine contains, besides the 30 kDa form, a new form of 45 kDa characterized by different glycosylation type and degree.

AB023, novel polyene antibiotics. II. Isolation and structure determination.

AB023, a complex of new polyene antibiotics, was isolated from a soil Streptomyces strain. The two main components, pentaene antibiotics AB023a and AB023b, were separated and purified by preparative chromatographic methods and their structures were determined by extensive NMR and mass spectrometric studies.

A42867, a novel glycopeptide antibiotic.

A42867 is a new glycopeptide antibiotic of the ristocetin-vancomycin class active against aerobic and anaerobic Gram-positive bacteria. A42867 is produced by a strain of Nocardia nov. sp. ATCC 53492. A42867 was isolated during a screening program aimed at the discovery of new members of this glycopeptide class of antibiotics, by affinity chromatography based on an acyl-D-alanyl-D-alanine probe. The structure of A42867 was elucidated by fast atom bombardment MS, high field 2D 1H NMR spectroscopy, and HPLC analysis of the hydrolyzed carbohydrates. A42867 differs from vancomycin in the sugar portion and in the presence of only one chlorine atom in the peptide core. Its biological activity on Gram-positive aerobic and anaerobic bacteria is similar to that of other antibiotics of this group.

Measured variation in boron loads reaching European sewage treatment works.

Per capita boron loads reaching 48 sewage treatment works (STWs) in The Netherlands, Germany, Italy, and the UK have been determined from monitoring data. These have been compared with the per capita input predicted from boron in detergents, as determined from detergent product sales data. The resulting distribution of the ratios of measured boron to boron predicted from consumer usage has a 90th percentile of less than 1.5. Boron has previously been shown to be a good marker for substances contained in detergent products, as it cannot be biodegraded and is not substantially adsorbed in the sewer, and there is little or no removal during sewage treatment processes. The monitoring information on the distribution of boron loads found at the different STWs should thus be indicative of the distribution of other substances released to the environment by detergent products, as specified by the relevant industrial category (IC 5-personal/domestic) in the Technical Guidance Documents. Variation in detergent product consumption figures from 18 European countries is also low, with the country with the highest per capita detergent consumption having only 1.3 times the European average detergent use. Thus the present practice of determining a "reasonable worst case" by multiplying the average per capita consumption by a factor of four to account for geographic differences in distribution, is considered to be inappropriate. This should be replaced by a factor of less than two, which combines within country and between country variations to provide a reasonable worst case approximation of the load reaching the sewage treatment facility.

GREAT-ER: a new tool for management and risk assessment of chemicals in river basins. Contribution to GREAT-ER #10.

The GREAT-ER (Geo-referenced Regional Exposure Assessment Tool for European Rivers) project team has developed and validated an accurate aquatic chemical exposure prediction tool for use within environmental risk assessment schemes. The software system GREAT-ER 1.0 calculates the distribution of predicted environmental concentrations (PECs) of consumer chemicals in surface waters, for individual river stretches as well as for entire catchments. The system uses an ARC/INFO-ArcView (ESRI) based Geographical Information System (GIS) for data storage and visualization, combined with simple mathematical models for prediction of chemical fate. At present, the system contains information for four catchments in Yorkshire, one catchment in Italy, and two in Germany, while other river basins are being added. Great-ER 1.0 has been validated by comparing simulations with the results of an extensive monitoring campaign for two 'down-the-drain' chemicals, i.e. the detergent ingredients boron and Linear Alkylbenzene Sulphonate (LAS). GREAT-ER 1.0 is currently being expanded with models for the terrestrial (diffuse input), air and estaurine compartments.