Bo Nilson

Purification of antibodies using protein L-binding framework structures in the light chain variable domain

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera

We previously discovered a symbiotic lactic acid bacterial (LAB) microbiota in the honey stomach of the honeybee Apis mellifera. The microbiota was composed of several phylotypes of Bifidobacterium and Lactobacillus. 16S rRNA gene sequence analyses and phenotypic and genetic characteristics revealed that the phylotypes isolated represent seven novel species. One grouped with Lactobacillus kunkeei and the others belong to the Lactobacillus buchneri and Lactobacillus delbrueckiisubgroups of Lactobacillus. We propose the names Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov. for these novel species, with the respective type strains being Fhon13NT ( = DSM 26257T = CCUG 63287T), Bin4NT ( = DSM 26254T = CCUG 63291T), Hon2NT ( = DSM 26255T = CCUG 63289T), Hma8NT ( = DSM 26256T = CCUG 63629T), Hma2NT ( = DSM 26263T = CCUG 63633T), Bma5NT ( = DSM 26265T = CCUG 63301T) and Biut2NT ( = DSM 26262T = CCUG 63631T).

Reactive Oxygen Species Produced by the NADPH Oxidase 2 Complex in Monocytes Protect Mice from Bacterial Infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47phox) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN+ mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b+Ly6G+ cells defined as neutrophils. MN+ mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN− mice. Most strikingly, MN+ mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN− mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.

On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins

Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement-inhibitory activity.

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (KD) for vitronectin binding to UspA2 was 2.3 × 10−8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a KD of 7.9 × 10−8 M. Electron microscopy verified that the active binding domain (UspA230–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA230–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.

Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid-phase radioimmunoassays

Using the newly described streptococcal surface protein, protein G, which has powerful immunoglobulin G binding properties, solid-phase radioimmunoassays were developed for the quantitation of goat and rat immunoglobulin G bound to the plastic surface of microtiter plates. The binding of goat immunoglobulin G to the surface via a specific antigen (guinea pig alpha 1-microglobulin) permitted the determination of antigen-specific antibodies with a detection limit of 50-100 ng. Optimum assay conditions were established and the whole assay procedure could be brought to completion at room temperature in less than a working day. The value of the assays was illustrated by monitoring rat and goat immunoglobulin G antibodies during their purification from whole sera by classical chromatographic procedures.

Cross-reacting monoclonal anti-α1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay

In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin.

Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems

Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Is a Sensitive and Specific Method for Identification of Aerococci

ABSTRACT Conventional methods for the identification of human-pathogenic aerococci to the species level are not reliable. We show that matrix-assisted laser desorption ionization–time of flight mass spectrometry correctly identifies aerococci to the species level and that it can be used to identify aerococci with high specificity in the diagnostic clinical microbiology laboratory.

Acoustic trapping for bacteria identification in positive blood cultures with MALDI-TOF MS.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.