Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.

Antibody repertoire diversity, potentially as high as 10(11) unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 x 10(6) reads, including more than 1.9 x 10(5) high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 x 10(4) sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 x 10(10). The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.

The role of echocardiography in the investigation of focal cerebral ischaemia.

One-hundred and ten patients referred for echocardiography to exclude a cardiac source of cerebral emboli were prospectively studied. Four patients with known cardiac abnormalities, for which they were receiving inadequate anticoagulation, were excluded from the study, and 18 patients were subsequently found to have a non-embolic cause for their cerebral pathology. Twenty-eight patients with a normal clinical examination, chest X-ray and electrocardiogram, and 27 patients with hypertension alone had echocardiograms which did not reveal a cardiac source of embolus. Of the remaining group of 33 patients, six were found to have a probable cardiac source of embolus and nine had abnormalities which may be associated with cerebral emboli. Echocardiography may not be indicated in patients with a normal clinical examination, chest X-ray and electrocardiogram, and in patients with hypertension alone. However, if these patients are excluded echocardiography gives a high yield of positive findings which may be of practical importance in the management of the patient.