Solid-phase enzyme immunoassay for rotavirus antigen: faecal protease activity as a reason for false-negative results.

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.

Effects of sublethal concentrations of antimicrobial agents on the hemagglutination, adhesion, and ultrastructure of pyelonephritogenic Escherichia coli strains.

The effects of trimethoprim, sulfadiazine, sulfamethoxazole, and sulfathiazole on the hemagglutination and adhesion by three Escherichia coli strains were studied. The strains were isolated from the urine of patients with acute pyelonephritis and carried P antigen-recognizing fimbriae (P fimbriae). At antimicrobial concentrations of 12.5 to 50% of the minimal inhibitory concentration, the ability of the bacteria to agglutinate human erythrocytes and to adhere to human buccal cells was markedly reduced. This reduction corresponded to a decrease in the number of P fimbriae per cell, suggesting that the antimicrobial agents decreased adhesion and hemagglutination by interfering with the formation of fimbriae. No major changes were observed in the outer membrane protein pattern of trimethoprim-treated cells, whereas freeze-fracture electron micrographs showed deorganization of both the cytoplasmic and outer membranes in bacteria opposed to sublethal concentrations of trimethoprim.

Time-resolved fluorescence imaging for quantitative histochemistry using lanthanide chelates in nanoparticles and conjugated to monoclonal antibodies.

Tissue and cell examinations have a potential to produce extremely valuable information about antigen quantities in samples. Using currently available methods, a truly quantitative analysis is nearly impossible. We have previously shown that immunohistochemical (IHC) detection of prostate-specific antigen and human glandular kallikrein from prostatic tissue, together with time-resolved fluorescence imaging (TRFI), is a suitable method for obtaining quantitative data from biological samples and that the signal response is linear. In this paper we show that Eu-chelate containing particles in the nanometer range are suitable labels for quantitative IHC. Even single nanoparticle molecules can be detected by TRFI and the signals measured can be readily quantitated. The signal intensity correlates very well with the amount of bound label, and the use of nanoparticles could markedly improve the sensitivity of quantitative IHC methods. TRFI provides a powerful tool for providing quantitative data about antigens or transcripts in tissue sections or cultured cells. It is also of major importance in standardization and optimization of protocols for fixation and tissue preparation, including antigen retrieval methods.

Escherichia coli strains binding neuraminyl alpha 2-3 galactosides.

A total of 46 E. coli strains showing mannose-resistant, P-blood-group independent hemagglutination of human erythrocytes were tested for binding to neuraminic acid. Nine of the strains completely lost their hemagglutination activity after the erythrocytes were treated with neuraminidase. To characterize the receptor structure, different neuraminic acid containing glycoproteins, their desialylated derivatives and neuraminyl oligosaccharides were tested for hemagglutination inhibition. These studies showed that the nine strains had binding specificity for alpha 2-3 linked neuraminic acid.

Production and characterization of novel anti-prostate-specific antigen (PSA) monoclonal antibodies that do not detect internally cleaved Lys145-Lys146 inactive PSA.

BACKGROUND: The nature of free, uncomplexed prostate-specific antigen (PSA) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen. METHODS: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies. RESULTS: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA. CONCLUSIONS: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.

Lipopolysaccharide, capsule, and fimbriae as virulence factors among O1, O7, O16, O18, or O75 and K1, K5, or K100 Escherichia coli.

K1, K5, and K100 Escherichia coli isolates of the lipopolysaccharide antigen types O1, O7, O16, O18, or O75, which had formerly been assigned to clonal groupings were compared with K? E. coli isolates and with laboratory-derived mutants defective in capsule or lipopolysaccharide synthesis. The amount of K1 capsule, the length distribution of the lipopolysaccharide, and the expression of type I and P fimbriae were determined. The clonal groupings were uniform with regard to these properties within each group but different from each other. Many of the K? strains differed from the clonal representatives. The results are interpreted with regard to the different diseases caused by each of these bacterial groups.

P-antigen-recognizing fimbriae from human uropathogenic Escherichia coli strains.

P-antigen-recognizing fimbriae (P fimbriae) from four pyelonephritogenic Escherichia coli strains and type 1 fimbriae from an E. coli strain and a Salmonella typhimurium strain were purified. The P fimbriae were morphologically similar to type 1 fimbriae. The purified P fimbriae agglutinated neuraminidase-treated human P1 and P2k erythrocytes but not p erythrocytes, which lack all P-blood group-specific glycosphingolipids. However, coating of neuraminidase-treated p erythrocytes with globoside rendered such erythrocytes agglutinable by the P fimbriae. The hemagglutinations were in all instances fully inhibited by the synthetic alpha-D-Galp-(1-4)-beta-D-Galp-1-O-Me glycoside. The binding specificity of the P fimbriae could also be demonstrated by using fimbriae coated onto latex particles and nontreated erythrocytes. It was thus concluded that the P fimbriae recognize and bind to the alpha-D-Galp-(1-4)-beta-D-Galp carbohydrate sequence occurring in the series of P-blood group antigen-specific glycosphingolipids. In contrast to both type 1 fimbriae, all four P fimbriae preparations showed multiple bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were raised in rabbits against the various E. coli fimbriae. In enzyme-linked immunosorbent assays each one of the antisera to the P fimbriae reacted to titers of log 4 to 7 with both the homologous and the heterologous P fimbriae, but not with the type 1 fimbriae of E. coli. In a reciprocal fashion, the antiserum to the type 1 fimbriae of one E. coli strain reacted only with the homologous type 1 but not with any of the P fimbriae preparations.

Characterization and processing of prostate specific antigen (hK3) and human glandular kallikrein (hK2) secreted by LNCaP cells.

Prostate specific antigen (PSA, hK3) in serum is predominantly complexed to alpha-1-antichymotrypsin (ACT), but a minor fraction remains in a free form despite the very large excess of serine protease inhibitors and alpha-2-macroglobulin. The fraction of free to total PSA is significantly lower in prostate cancer (CaP) compared to benign prostatic hyperplasia (BPH) which provides improved discrimination of these conditions. The molecular nature of free PSA in the circulation and the reason for its varying concentration in malignant and benign conditions is currently not known. The objective of the present investigation was to study the secretion of PSA and human glandular kallikrein 2 (hK2) by the LNCaP prostate cancer cell line, and to purify and characterize both proteins. LNCaP PSA was thoroughly characterized by immunological characterization, SDS-PAGE, isoelectric focusing, gel filtration, aminoterminal sequencing, reverse-phase chromatography, mass spectrometry and enzymatic activity measurements. LNCAP cells produced approximately equal amounts of zymogen (proPSA) and the one-chain mature form of PSA, whereas there was no evidence for the secretion of any internally cleaved forms. LNCaP cells secreted hK2 into the growth medium at about 3-5% of the amount of PSA. One-chain, mature PSA and hK2 obtained when LNCaP cells were grown in the presence of fetal bovine serum, had no enzymatic activity, but were active when the cells were grown in the absence of serum. Using enzymatically active recombinant hK2, it was possible to activate proPSA secreted by LNCaP cells. ProPSA formed two bands with high isoelectric points (8.2 and 8.4), which disappeared when proPSA was converted to mature PSA with hK2. Cancerous cells produce the zymogen forms of PSA, which by their isoelectric pI points seem to be found in serum of prostate cancer patients, but not BPH patients. Mature, one-chain PSA is inactive in the presence of serum. These findings may be highly relevant for the understanding of the generation of free and complexed PSA in the circulation.

Association of P and other fimbriae with clinical pyelonephritis in children.

196 episodes of urinary tract infection in children were analysed. 74 were classified as pyelonephritic (PN), 61 as cystitis (C), and 61 as asymptomatic bacteriuria (ABU) on the basis of three clinical signs (elevated temperature, erythrocyte sedimentation rate, and/or white blood cell count). The frequency of P-fimbriae was found high in PN (77%), and significantly lower in C (23%), ABU (20%) and among fecal strains (16%). The common, type 1 fimbriae were also more frequent in PN (92%) than in the other groups (84-76%), whereas other, so-called X-fimbriae, were relatively rare in all the patient groups (15-6%). P-fimbriation was not significantly associated with the presence or absence of reflux or obstructive anomalies. By contrast, the frequency of P-fimbriation increased with increasing severity of the clinical symptoms of pyelonephritis (95% in episodes of elevated temperature, erythrocyte sedimentation rate and white blood cell count).

The role of pili in the adhesion of Escherichia coli to human urinary tract epithelial cells.

Pili or fimbriae were purified by a new technique involving solubilization from the bacterial outer membrane by deoxycholate and separation from flagella by 6M urea. This technique was employed to clarify the role of pili for the adherence of urinary tract pathogenic E. coli; a virulence factor in urinary tract infection. The isolated pili formed single bands in SDS gels and were pure by serologic criteria. They retained the binding properties of the whole piliated bacteria, since they bound to uroepithelial cells and agglutinated erythrocytes. Antibodies to purified pili blocked adhesion. The adhesion and hemagglutination reactions by the strains used for pilus purification were mannose-resistant but globotetraos-sensitive, i.e. the strains recognized globoseries glycolipid receptors in the target cells. The occurrence of this property in a freshly collected material of strains was tested using erythrocytes of blood groups P1, P2k and p.

Mannose-resistant haemagglutination and P antigen recognition are characteristic of Escherichia coli causing primary pyelonephritis.

Thirty-two Escherichia coli strains from 30 children with pyelonephritis were examined for their haemagglutination patterns and O and K serotypes. 29 (91%) of the strains showed mannose-resistant haemagglutination (MRHA). By use of well-defined target cells, these MRHA+ strains could be shown to recognise human cells either in a P-specific manner (recognising a specific galactosyl-galactose structure which is part of P blood groups antigens) or in a separate, X-specific manner. Both recognition mechanisms could occur separately or together on the same bacteria, the frequencies of P and X specificity being 81 and 19%, respectively. Both MRHA and P specificity were significantly associated with the O antigens 01, 04, 06, 016, and 018, and the capsular antigen K1, which have previously been associated with pyelonephritis. However, the association of MRHA and P specificity with upper urinary tract infection in children is greater than that of any other laboratory-defined bacterial characteristic.