The FlowMetrixTM System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95®-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins.

The proteins of the outer membrane of Neisseria gonorrhoeae play an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med. 143:741-758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3'-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.

Chronic kidney disease (CKD) occurs frequently after liver transplantation (LT) and is associated with significant morbidity and mortality. Thus, there is a pressing need to identify characteristics and biomarkers diagnostic of CKD to enable early diagnosis allowing preemptive interventions, as well as mechanistic insights into the progression from kidney injury to irreversible kidney failure. We analyzed 342 patients who had baseline GFR>60 at time of LT and are now >3 years post-LT. Risk factors for post-LT CKD were compared between 3 different groups defined by current GFR: >90 (n=40), 60–90 (n=146) and <60 (n=156) ml/min. Age, cyclosporine use, and pre-LT GFR were independently associated with new onset CKD. A subset (n=64) without viral/immune disease or graft dysfunction underwent multi-analyte plasma proteomic evaluations for correlation with CKD. Plasma proteomic analysis of two independent cohorts, test (n=22) and validation (n=42), identified 10 proteins highly associated with new onset CKD. In conclusion, we have identified clinical characteristics and a unique plasma proteomic signature correlating with new onset CKD after LT. These preliminary results are currently being validated in a prospective, multi-center study to determine if this signature precedes the onset of CKD and resolves with early interventions aimed at preserving kidney function.

The attachment of gonococci to mucosal surfaces is of primary importance in their interactions with the genito-urinary tract of the human host. This process may be, in part, mediated by pili, which are proteinaceous appendages emanating from the bacterial cell surface. Observations by Kellogg et al. (1) suggested that certain colonial morphological phenotypes are associated with virulence. Subsequently, Swanson et al. (2) demonstrated that gonococci giving rise to the colonial phenotype associated with virulence carry pili on their surface. Subsequently, piliated gonococci have been shown to agglutinate erythrocytes (3), and attach to spermatozoa (4), epithelial cells (5), and human fallopian tubes in organ culture (6, 7).Pili are filaments, 1-4 ~m in length, composed of repeating, identical subunits of the protein pilin. Pilin demonstrates a variable apparent molecular weight, depending on type, of 17,500-21,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8). Gonococcal pilins possess a single intramolecular disulfide bond and two methionine residues, which allows for cleavage by cyanogen bromide into three peptide fragments (CB-1, 2, and 3) (9). The CB-2 fragment (residues 8-92 in strain MS11 pilin) possesses the erythrocyte-binding domain, as evidenced by its ability to compete with intact pili in a hemagglutination assay. Peptide mapping of CB-2 fragments from serologically distinct pilin (strains MS11 and R10) demonstrates homology in this area of the protein (9). The complete amino acid sequence of MS 11 pilin has been obtained, as well as the first 59 amino acids of R10 pilin (10). Comparison of these sequences demonstrates identity through amino acid 59, providing additional evidence that the amino-terminal half of gonococcal pilin is highly conserved. Conversely, peptide mapping of CB-3 fragments (residues 93-159 in MSll pilin) demonstrates significant differences between pilin types, indicating that the carboxy-terminal portion of the protein contains the variable domain (9).The distinct serological heterogeneity of gonococcal pili has been demonstrated by a number of investigators (8, 11). Immunization of rabbits and mice with purified pili elicits immune responses that are strikingly type specific (8). Vaccination of humans with purified pili produced primarily type-specific anti-