AP200 Sigma-AldrichGoat Anti-Mouse light chain Antibody

In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

Streptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes.The genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated φSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of φSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to φSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. φSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain.The sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and φSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.

A hydrogen peroxide (H(2)O(2))-enhanced iron (Fe(0))-mediated aeration (IMA) process has been recently demonstrated to effectively remove organic wastes from mature landfill leachate. In this paper, the kinetics and oxidative mechanisms of the enhanced IMA treatment were studied. Bench-scale full factorial tests were conducted in an orbital shaker reactor for treatment of a mature leachate with an initial chemical oxygen demand (COD) of 900-1200 mg/L. At the maximum aeration rate (8.3 mL air/min mL sample), process variables significantly influencing the rates of H(2)O(2) decay and COD removal were pH (3.0-8.0), initial H(2)O(2) doses (0.21-0.84 M), and Fe(0) surface area concentrations (0.06-0.30 m(2)/L). Empirical kinetic models were developed and verified for the degradation of H(2)O(2) and COD. High DO maintained by a high aeration rate slowed the H(2)O(2) self-decomposition, accelerated Fe(0) consumption, and enhanced the COD removal. In hydroxyl radical (OH*) scavenging tests, the rate of removal of glyoxylic acid (target compound) was not inhibited by the addition of para-chlorobenzoic acid (OH* scavenger) at pH 7.0-7.5, ruling out hydroxyl radical as the principal oxidant in neutral-weakly basic solution. These experimental results show that this enhanced IMA technology is a potential alternative for the treatment of high strength recalcitrant organic wastewaters.

BACKGROUND: It has been shown that a vacuum cleaner (VC) can increase airborne cat allergen levels. This study aimed to compare the degree of leakage of airborne Fel d 1 levels among five different VCs, both under laboratory conditions and in an apartment with cats. METHODS: Three of the VCs were marketed as antiallergic: a HEPA filter VC (VC A), a water impingement and HEPA filter VC (VC B), and a foam fabric filter VC (VC C). The other two were standard VCs: VC D and VC E. VCs were tested in a 20 m3, airtight, experimental room and in a 53 m3 living room in an apartment with three cats. Air was sampled with a glass-fiber filter and an impinger at 20 l/min for 30 min before, during, and after vacuuming. Airborne Fel d 1 was measured with a two-site monoclonal ELISA assay. RESULTS: In the experimental room, no airborne Fel d 1 level was measured before using the VCs. After introducing a dust sample containing Fel d 1 in the VCs, we found that VCs A, B, and E did not provoke any increase in airborne Fel d 1. In contrast, VCs C and D significantly increased airborne Fel d 1 levels (GM: 4.9 and 5.3 ng/m3, respectively). In the apartment, all VCs induced an increase in airborne Fel d 1, which was carried by particles greater than 5 microm. However, VCs C and D provoked significantly greater increases in airborne Fel d 1 than VCs A, B, and E (P=0.0001). CONCLUSIONS: Our results suggest that: 1) The two VCs with leakage in the experimental room had greater leakages in the apartment. 2) In the apartment with cats, all VCs provoked increases in airborne Fel d 1, primarily carried by large particles. 3) Given the increased marketing of antiallergic VCs, further studies are needed to standardize methods for testing airborne allergen leakage by VCs.