Phage-based molecular directed evolution yields multiple tandem human IgA affibodies with intramolecular binding avidity.

Affibodies are a group of affinity proteins that are based on a 58-amino-acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. A single human IgA affibody with high IgA affinity has been generated by directed evolution. It remains interesting whether tandem IgA affibody proteins could increase binding capacity. Here, we report the generation of multiple tandem IgA affibodies by directed evolution using a combinatorial phage library displaying the IgA affibody A1 and/or A2 linked with three random amino acids. These affibodies exhibited markedly increased IgA binding capacity, as shown by enzyme linked immunosorbent assay, immunoblotting and surface plasmon resonance assays. We further showed that these tandem IgA affibodies displayed preferential binding to intact IgA molecules compared to individual IgA chain, suggesting intramolecular binding avidity. Our data demonstrates that artificial multiple tandem human IgA affibodies with relevant biological binding avidity were successfully yielded by phage-based molecular evolution. These results have broad implications for the design and development of binding proteins that target important biological molecules.

[Gastrointestinal dysfunction in acute severe mountain sickness and its relation with multiple organ dysfunction syndrome].

OBJECTIVE: To investigate the relationship between gastrointestinal dysfunction (GD) and multiple organ dysfunction syndrome (MODS) in acute severe mountain sickness (ASMS), including high altitude pulmonary edema (HAPE), and high altitude cerebral edema (HACE), by a retrospective study of medical records and prospective study of hospitalized patients. METHODS: In retrospective study, the clinical data of 3 184 inpatients of General Hospital of Tibetan Military Command suffering from ASMS in the past 50 years (from June, 1958 to June, 2007) were collected. Statistical analysis was performed to study the relationship between GD and MODS in these patients. For the prospective study, 10 admitted patients of ASMS were included. Gastroscopic examination was performed for the ASMS patients, and gastric and duodenal mucosa was scrutinized. At the same time, 30 g of glutamine (Gln) capsule was orally ingested each day for 3 days after the first day of admission. Ten healthy volunteers were included in the control group, and received the same treatment. The levels of serum diamine oxidase (DAO), malonic dialdehyde (MDA), endotoxin and lactulose/mannitol (L/M) ratio were detected before and after treatment in two groups. RESULTS: First, 49.8% of the patients with ASMS were complicated with GD, with 1.5% of fairy stool, and 1.0% with occult blood in stool. In 83 cases of ASMS complicated with MODS, 21.7% (18 cases) appeared GD, and the score of GD was 5.5 in the total score of all organ injury. Second, endoscopic examination showed extensive edema and localized hemorrhage in gastrointestinal mucous membrane, with dotted and patched erosion in gastric antrum and fundus. The pre-treatment DAO, MDA, and endotoxin were higher in the observation group than those in the control group (all P<0.01). After 3 days of Gln capsule treatment, DAO, MDA, and endotoxin were significantly decreased in the observation group (P<0.05 or P<0.01). The pre-treatment L/M ratio in observation group was significantly higher than that in healthy control group (150.69+/-19.91 vs. 117.91+/-17.78, P<0.01). The L/M ratio was significantly decreased after the treatment, as it decreased to 129.37+/-19.75 (P<0.05). However, no significant change in the healthy control group was observed. CONCLUSION: GD plays a major role in the pathogenesis of MODS in ASMS patients.

Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig)-binding proteins (IBPs) with four kinds of Ig molecules.

BACKGROUND: Protein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting. RESULTS: We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively. CONCLUSION: In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.