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1.  Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis 
Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.
Author Summary
Burkholderia pseudomallei is an environmental bacterium and the cause of melioidosis. Culture of patient samples is the “gold standard” diagnostic test, but may take up to 7 days to complete. Melioidosis has a 10–40% case fatality rate depending on the geographic location. Delays in diagnosis could lead to administration of ineffective antimicrobial therapy, since B. pseudomallei is resistant to empiric antibiotic regimens. Therefore, we have developed a lateral flow immunoassay that can be used in the clinical setting to diagnose melioidosis in 15 minutes. The test promises to provide improved management of patients with melioidosis.
doi:10.1371/journal.pntd.0002727
PMCID: PMC3961207  PMID: 24651568
2.  Application of a Multigene RT-PCR assay for the detection of Mammaglobin and complementary transcribed genes in breast cancer lymph nodes 
Clinical chemistry  2002;48(8):1225-1231.
Background: Mammaglobin mRNA expression is found in 70-80 % of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P and GABAπ complement the expression of mammaglobin. Collectively the expression profile of these four genes could be utilized as a diagnostic and prognostic indicator for breast cancer.
Methods: A multigene RT-PCR assay was established to detect the expression of mammaglobin, GABAπ, B305D and B726P simultaneously. Specific primers and Taqman® probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens.
Results: The multigene RT-PCR assay detected significant expression signals in 27/27 primary and in 50/50 lymph-node-metastatic breast tumor samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various normal tissues or in 14 colon tumor samples.
Conclusion: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.
PMCID: PMC1482782  PMID: 12142378
3.  Burkholderia pseudomallei Proteins Presented by Monocyte-Derived Dendritic Cells Stimulate Human Memory T Cells In Vitro▿  
Infection and Immunity  2010;79(1):305-313.
Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4+ T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8+ T cells. Activation of antigen-specific CD4+ T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.
doi:10.1128/IAI.00803-10
PMCID: PMC3019888  PMID: 21041491
4.  Lateral Flow Immunoassay for Diagnosis of Trypanosoma cruzi Infection with High Correlation to the Radioimmunoprecipitation Assay▿  
The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) “gold standard” in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a specificity of 99.1%.
doi:10.1128/CVI.00383-08
PMCID: PMC2668284  PMID: 19211772
5.  Expression of Mammaglobin, B305D, GABAπ and B726P and elevation of Mammaglobin protein in the peripheral blood of women with untreated breast cancers 
Clinical chemistry  2004;50(11):2069-2076.
Purpose
The aim of this study was to analyze the utility of a mammaglobin multigene RT-PCR assay and a mammaglobin sandwich ELISA to detect peripheral blood samples of breast cancer patients.
Experimental Design
Peripheral blood samples of 147 untreated Senegalese women with biopsy confirmed breast cancer were collected. The samples were tested for mammaglobin and 3 breast cancer associated gene transcripts using a multigene real-time RT-PCR assay and for secreted mammaglobin protein using a sandwich ELISA format. Patient information regarding demographic and clinical staging of disease was also collected.
Results
In 77 % of the breast cancer blood samples a positive expression signal was found using the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. 50 samples from healthy female donors tested negative. Circulating mammaglobin protein was found in 68 % of the breast cancer sera, whereas 38 % showed significantly elevated protein levels in comparison to a mixed control population. Statistical correlations were found between the detection of mammaglobin protein in serum, presence of mammaglobin mRNA expressing cells in blood, stage of disease and tumor size.
Conclusions
The multigene mammaglobin RT-PCR assay and mammaglobin sandwich ELISA could be valuable tools to detect metastatic disease and to monitor therapeutic efficiency. Both assays together provided a diagnostic sensitivity of 83 %. Use of the multigene RT-PCR increased detection sensitivity from 61 to 77 % in comparison to mammaglobin expression alone.
doi:10.1373/clinchem.2004.038687
PMCID: PMC1482781  PMID: 15375015
multigene RT-PCR; circulating tumor cell detection; breast cancer
6.  Identification and Characterization of Putative Secreted Antigens from Babesia microti 
Journal of Clinical Microbiology  2003;41(2):723-729.
The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.
doi:10.1128/JCM.41.2.723-729.2003
PMCID: PMC149722  PMID: 12574273
7.  Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis 
Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ∼98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ∼93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.
doi:10.1128/CDLI.9.4.883-891.2002
PMCID: PMC120014  PMID: 12093690
8.  Serological Expression Cloning and Immunological Evaluation of MTB48, a Novel Mycobacterium tuberculosis Antigen 
Journal of Clinical Microbiology  2001;39(7):2485-2493.
Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.
doi:10.1128/JCM.39.7.2485-2493.2001
PMCID: PMC88174  PMID: 11427558
9.  Serodiagnosis of Human Granulocytic Ehrlichiosis by Using Novel Combinations of Immunoreactive Recombinant Proteins 
Journal of Clinical Microbiology  2001;39(7):2466-2476.
A panel of seven recombinant antigens, derived from Ehrlichia phagocytophila (the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (ank). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of E. phagocytophila infection.
doi:10.1128/JCM.39.7.2466-2476.2001
PMCID: PMC88172  PMID: 11427556
10.  Mass Spectrometric Identification of Mtb81, a Novel Serological Marker for Tuberculosis 
Journal of Clinical Microbiology  2000;38(6):2354-2361.
We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV+ TB+ sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB.
PMCID: PMC86802  PMID: 10835002
11.  Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti 
Infection and Immunity  2000;68(5):2783-2790.
Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.
PMCID: PMC97488  PMID: 10768973

Results 1-11 (11)