Journal of Proteome Research and serodiagnostic antigens and to classify the reactive antigens based on functional and physical properties. Protein microarrays can be used with relative ease to probe the entire proteome of diﬀerent infectious microorganisms including bacteria, viruses and parasites.3 13 This approach permits assessing the repertoire of antibodies produced in response to infections or vaccinations from large collections of individual patient sera, and can be used to perform large-scale sero-epidemiological and sero- surveillance analyses not possible with other technologies, while consuming small quantities (<2 μL) of each serum sample. Probing large numbers of patient specimens empowers the statistical tests resulting in more reliable conclusions while correcting for false discovery required for multiple comparison testing inherent in microarray analysis.3,5,7,9 Moreover, microarrays can display all proteins of an infectious agent and may allow for identiﬁcation of novel antigens, otherwise undetectable by methods like 2-D gels that are highly biased by microbial protein expression patterns. Brucellosis is a worldwide zoonosis caused by bacteria of the genus Brucella. Brucella melitensis, the most virulent species infecting humans, also infects goats, sheep and cattle,14 16 in Central and South America, the Middle East, East Asia, and some southern European countries.17,18 Consumption of animal products, direct animal contact and inhalation of aerosolized bacteria in the laboratory setting (indicating the potential for air-borne spread via a bioterrorism-type attack) are well-recognized routes of infection. The current knowledge of protein antigens recognized by humans and reservoir animals is based on limited numbers of studies on Brucella melitensis and Brucella abortus.19 31 We recently constructed a pilot proteome array consisting of 1406 B. melitensis proteins and observed marked diﬀerences in immune responses between humans and goats.5 Here we have expanded our previous study to the full proteome microarray consisting of 3046 B. melitensis proteins, by including expression of newly cloned 1640 Bm genes. In addition to developing a better classiﬁer capable of predicting disease based on serological response, more importantly, this study allows an estimate of the extent of immune reactivity against the whole proteome, and prediction of proteomic features that may dictate immune recogni- tion for other yet uncharacterized gram-negative bacteria. ’ MATERIALS AND METHODS Ethics Statement Human sera were obtained from patients enrolled in a prospec- tive clinical study of brucellosis in Lima, Peru. The human subjects part of the study was approved by the Humans Research Protections Committee of the University of California San Diego, the Comite de Etica of Universidad Peruana Cayetano Heredia, Lima, Peru and the Comite de Etica of Asociacion Beneﬁca PRISMA, Lima, Peru, all of whom have maintained federal wide assurances with the United States Department of Health and Human Services. All patients provided written informed consent prior to enrollment in the study, and signed consent forms have been stored in locked ﬁles in study oﬃces at UPCH and AB PRISMA, Lima, Peru. Human Serum Samples Human sera were obtained from patients in Lima, Peru. All patients in this studywere infected with B. melitensis biovar 1. Forty- two patients were conﬁrmed to have acute brucellosis by positive culture, positive Rose Bengal test and by tube agglutina- tion tests titers g1/160. Eighteen patients presenting with brucellosis-compatible syndromes were culture negative and Rose ARTICLE Bengal positive, and treated according to standard antibiotic therapy within 2 days of serum sampling. Additional control patient samples included 13 sera from Rose Bengal-negative patients, 44 samples from ambulatory healthy controls from north Lima where brucellosis occasionally aﬀects patients, and sera from humans in the U.S. where brucellosis is not found. Brucella melitensis Lipopolysaccharide Purification Approximately 10 g of autoclaved pellet of B. melitensis 16 M were used to isolate LPS using the hot phenol-water method.32 The puriﬁed LPS was treated with RNase, DNase and Protei- nase-K, and the hot phenol-water treament was repeated. B. melitensis LPS obtained from both upper phenol saturated aqu- eous layer (aqueous phase) and lower water saturated phenol layer (phenol phase) were pooled. LPS from E. coli 055:B5 was purchased from Sigma Chemicals (St. Louis, MO). The LPS from E. coli 055:B5 and B. melitensis 16 M were analyzed on a gradient 4 12% Tris-Glycine sodium dodecyl sulfate (SDS) polyacrylamide gel (Invitrogen Corp., Carlsbad, CA) under redu- cing conditions. The presence of LPS in the gels was detected with a periodic acid silver stain33 and protein using Coomassie blue stain. B. melitensis LPS was quantiﬁed using a colorimetric assay to measure 2-keto-3-deoxyoctonate (KDO) concentration.34 E. coli 055: B5 LPS (Sigma, St. Louis, MO) was used as standard. Microarray Fabrication, Probing and Immunostrips Probing All ORFs from Brucella melitensis 16 M genomic DNA were identiﬁed using GenBank NC_003317 and NC_003318, and 1640 ORFs that were absent from pilot chip were ampliﬁed and cloned using a high-throughput PCR and recombination cloning method described previously.5 Microarrays and immunostrips were fabricated and probed as described before.5 Plasmids were expressed at 24 C for 16 h in in vitro transcription/translation E. coli reactions (Expressway Maxi kits from Invitrogen), accord- ing to the manufacturer’s instructions. For microarrays, 10 μL of reaction was mixed with 3.3 μL 0.2% Tween 20 to give a ﬁnal concentration of 0.05% Tween 20, and printed onto nitrocellu- lose coated glass FAST slides (Whatman) using an Omni Grid 100 microarray printer (Genomic Solutions). For immunostrips, B. melitensis LPS was printed at 0.01 mg/mL. All antigens were printed on Optitran BA-S 85 0.45 μm Nitrocellulose membrane (Whatman) using BioJet dispenser (BioDot) at 1 μL/cm, and cut into 3 mm strips. Human sera samples were diluted to 1:200 with 10 mg/mL E. coli lysate (Mclab). Microarray slides were in- cubated in biotin-conjugated secondary antibody (Jackson Im- munoResearch) diluted 1/200 in blocking buﬀer, and detected by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences). The slides were washed and air-dried by brief centrifugation. Microarray slides were scanned and analyzed using a Perkin-Elmer ScanArray Express HT microarray scanner. Intensities were quantiﬁed using QuantArray software. All signal intensities were corrected for spot-speciﬁc background. For immunostrips probing, human sera were diluted to 1/200 in 5% BSA solution containing 20% E. coli lysate (McLab). Strips were then incubated in alkaline phosphatase conjugated donkey antihuman immunoglobulin (anti-IgG, Fcγ fragment- speciﬁc, Jackson ImmunoResearch) secondary antibody, diluted to 1/2000, and reactive bands were visualized by incubating with 1-step Nitro-Blue Tetrazolium Chloride/5-Bromo-4-Chloro-30- Indolyphosphate p-Toluidine Salt (NBT/BCIP) developing buﬀer (Thermo Fisher Scientiﬁc). Immunostrips were scanned with Hewlett-Packard document scanner, and were quantiﬁed using Image J software. 4814 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824