Display of a peptide mimotope on a crystalline bacterial cell surface layer (S-layer) lattice for diagnosis of Epstein-Barr virus infection

Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72/p2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein-Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 individual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.     

Increased fibroblast growth factor-like autoantibodies in serum from a subset of patients with cancer-associated hypercalcemia

Basic fibroblast growth factor (bFGF) is a potent tumor angiogenesis factor which lacks an amino-terminal signal sequence and does not normally circulate in serum from normal subjects. Naturally-occurring autoantibodies which mimicked basic fibroblast growth factor were described in serum from patients with multiple endocrine neoplasia type 1 prolactinoma or sporadic growth-hormone-secreting adenoma associated with increased bFGF. Since bFGF was increased in serum from a variety of cancers, we used endothelial cell proliferation assay(s) to test for bioactivity in the IgG fraction of serum from 56 patients with cancer-associated hypercalcemia, and normal or control subjects. We now report increased IgG-like endothelial cell activity in serum from a hyper prolactinemic subset (4/19 breast cancer; 1/14 renal cancer; 0/23 lung cancer) of cancer-associated hypercalcemic subjects. Highest activity was found in serum from three breast cancer patients who suffered spinal cord compression/metastases. The activity had properties of antiidiotype bFGF antibodies including reaction with anti-human IgG antibodies, and complete neutralization by rabbit antibodies to intact bFGF. The activity in endothelial cells persisted after storage at 0-4 C for 5 yrs; and [prepared by SDS-PAGE and immunoblotting with anti-human IgG] had apparent mol wt corresponding to the heavy chains of IgG. Serum IgG-like activity from 5 of 5 breast cancer patients and 2 of 2 prostate cancer subjects tested [prepared by anti-bFGF antibody, protein-A immunoaffinity, and hydroxyapatite (HA) chromatography] yielded peak HA-adsorbed activity that eluted with 0.4 M sodium phosphate, and was neutralized 70% by antibodies to intact bFGF. Cancer sera mean peak specific activity (12.0 ng-eq bFGF/ug protein) (n = 7) significantly exceeded (P < 0.001) normal sera mean peak specific activity (0.46 ng-eq bFGF/ug protein) (n = 6) in the 0.4 M sodium phosphate eluate fraction from hydroxyapatite columns. These results imply that long-lasting, bioactive FGF-like autoantibodies may arise spontaneously (and contribute to pathophysiology) in subsets of cancer patients with osseous metastases.     

A microfluidic biosensor based on competitive protein adsorption for thyroglobulin detection

We report a microfluidic sensing platform for the detection of thyroglobulin (Tg) using competitive protein adsorption. Serum Tg is a highly specific biomarker for residual thyroid tissue, recurrence and metastases after treatment for differentiated thyroid cancer (DTC). Conventional Tg detection techniques require complicated immobilization of antibodies and need to form a sandwich assay using additional secondary antibodies to enhance the sensitivity. We present a fundamentally different sensing technique without using antibody immobilization on a microfluidic platform. We engineer two surfaces covered by two known proteins, immunoglobulin G (IgG) and fibrinogen, with different affinities onto the surfaces. The microfluidic device offers a selective protein sensing by being displaced by a target protein, Tg, on only one of the surfaces. By utilizing the competitive protein adsorption, Tg displaces a weakly bound protein, IgG; however, a strongly bound protein, fibrinogen, is not displaced by Tg. The surface plasmon resonance (SPR) sensorgrams show that five human serum proteins, albumin, haptoglobin, IgG, fibrinogen and Tg, have different adsorption strengths to the surface and the competitive adsorption of individuals controls the exchange sequence. The adsorption and exchange are evaluated by fluorescent labeling of these proteins. Tg in a protein mixture of albumin, haptoglobin, and Tg is selectively detected based on the exchange reaction. By using the technique, we obviate the need to rely on antibodies as a capture probe and their attachment to transducers.     

A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.     

Time-resolved fluoroimmunoassay of steroid hormones

The use of europium chelates as labels in immunoassays and their sensitive quantitation based on time-resolved fluorescence is reviewed. The technique is applied on competitive solid-phase immunoassays for direct determination of progesterone and estradiol in serum samples. Both antigen- and antibody-labelled competitive assays are described. The nonisotopic label technology, which provides a very high specific activity, as well as the antibody-labelled competitive assays, present several advantages in the assay of haptens as e.g. steroids. As the optimal sensitivity of competitive methods is not limited by the specific activity of the label the steroid assays which employ europium chelates as labels do not show any marked increase in sensitivity as compared to that achieved by using 125I. The potential sensitivity provided by the high specific activity of the label is optimally utilized in noncompetitive immunometric assays.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Elevated CRB3 expression suppresses breast cancer stemness by inhibiting β‐catenin signalling to restore tamoxifen sensitivity

Tamoxifen is a first‐line drug for hormone therapy (HT) in oestrogen receptor‐positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for β‐catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of β‐catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting β‐catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.     

SARS冠状病毒Mc区原核表达及包涵体复性的研究

构建SARS冠状病毒M蛋白膜内区(Mc蛋白)基因(Mc区)融合表达载体,表达并复性该蛋白。克隆Mc区,构建融合表达载体pET-32a(+)/Mc,融合表达Mc蛋白。纯化表达蛋白,采用逐步透析、降低蛋白浓度、加入氧化还原剂复性融合蛋白。用复性蛋白免疫兔产生抗血清,复性蛋白与SARS患者血清和健康人血清反应鉴定复性结果。结果显示,复性蛋白免疫家兔后产生抗血清,该复性蛋白与SARS患者血清特异结合,表明已成功复性该表达蛋白。为SARS疫苗和诊断试剂的研制奠定基础。     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Solid-phase synthesis of methyl carboxymycobactin T 7 and analogues as potential antimycobacterial agents

A solid-phase approach for the total synthesis of methyl carboxymycobactins 1a-d, with an on-resin cyclization leading to azopine 5 as the key step, was developed. The iron-affinity of these compounds was assessed by a competitive sulfoxine-binding assay, and antimycobacterial activity was tested against the growth of Mycobacterium avium.     

Urinary Nucleosides as Biomarkers of Breast,Colon, Lung,and Gastric Cancer in Taiwanese

Urinary nucleosides are associated with many types of cancer. In this study, six targeted urinary nucleosides, namely adenosine, cytidine, 3-methylcytidine, 1-methyladenosine, inosine, and 2-deoxyguanosine, were chosen to evaluate their role as biomarkers of four different types of cancer: lung cancer, gastric cancer, colon cancer, and breast cancer. Urine samples were purified using solid-phase extraction (SPE) and then analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Mann-Whitney U test and Principal Component Analysis (PCA) were used to compare differences in urinary nucleosides between patients with one of four types of cancer and healthy controls. The diagnostic sensitivity of single nucleosides for different types of cancer ranged from 14% to 69%. In contrast, the diagnostic sensitivity of a set of six nucleosides ranged from 37% to 69%. The false-positive identification rate associated with the set of six nucleosides in urine was less than 2% compared with that of less than 5% for a single nucleoside. Furthermore, combining the set of six urinary nucleosides with carcinoembryonic antigen improved the diagnostic sensitivity for colon cancer. In summary, the study show that a set of six targeted nucleosides is a good diagnostic marker for breast and colon cancers but not for lung and gastric cancers.     

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines.

A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell-surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth-promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell-conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2

Trastuzumab, a humanized antibody to HER-2, has been shown to be effective in the treatment of breast cancer in which HER-2 overexpression and metastasis occurs. In our search for an effective mimic epitope of HER-2 binding with trastuzumab and to develop HER-2 peptide vaccine, we screened a phage display 12-mer peptide library with trastuzumab as the target. A mimetic peptide (mimotope) H98 (LLGPYELWELSH) that could specifically recognize trastuzumab was isolated. The DNA encoding peptide H98 was cloned and expressed as the fusion protein GST-H98 in Escherichia coli BL21. The purified GST-H98 could specifically bind to trastuzumab and block the binding of trastuzumab to HER-2 protein. Moreover, H98 could significantly block the function of trastuzumab inhibiting the growth of cancer cells. Mice that were immunized with GST-H98 made specific antibody to H98 as well as to HER-2. In addition, T-cell proliferation occurred in mice immunized with GST-H98. Although no sequence homology was found between H98 and HER-2, through the use of structure analysis we were able to determine that peptide H98 contributed to a conformational epitope of HER-2. Furthermore, we determined that the last two amino acids at the C terminus, and the third together with the fourth amino acid at the N terminus of peptide H98 are critical to the binding of H98 to trastuzumab. As a result, we conclude that peptide H98 has potential for being developed as a HER-2 vaccine for biotherapy of cancer with HER-2 overexpression.     

Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer: a case-control study

Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.     

Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

The ATM gene and breast cancer: is it really a risk factor?

The genetic determinants for most breast cancer cases remain elusive. Whilst mutations in BRCA1 and BRCA2 significantly contribute to familial breast cancer risk, their contribution to sporadic breast cancer is low. In such cases genes frequently altered in the general population, such as the gene mutated in Ataxia telangiectasia (AT), ATM may be important risk factors. The initial interest in studying ATM heterozygosity in breast cancer arose from the findings of epidemiological studies of AT families in which AT heterozygote women had an increased risk of breast cancer and estimations that 1% of the population are AT heterozygotes. One of the clinical features of AT patients is extreme cellular sensitivity to ionising radiation. This observation, together with the finding that a significant proportion of breast cancer patients show an exaggerated acute or late normal tissue reactions after radiotherapy, has lead to the suggestion that AT heterozygosity plays a role in radiosensitivity and breast cancer development. Loss of heterozygosity in the region of the ATM gene on chromosome 11, has been found in about 40% of sporadic breast tumours. However, screening for ATM mutations in sporadic breast cancer cases, showing or not adverse effects to radiotherapy, has not revealed the magnitude of involvement of the ATM gene expected. Their size and the use of the protein truncation test to identify mutations limit many of these studies. This latter parameter is critical as the profile of mutations in AT patients may not be representative of the ATM mutations in other diseases. The potential role of rare sequence variants within the ATM gene, sometimes reported as polymorphisms, also needs to be fully assessed in larger cohorts of breast cancer patients and controls in order to determine whether they represent cancer and/or radiation sensitivity predisposing mutations.     

High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.     

Development of a homogeneous high throughput fluorescence polarization assay for G protein-coupled receptor binding

Traditional methods that follow receptor ligand interactions are competitive assays in which the test compound displaces a radiolabeled molecule. These assays require either a time-consuming step for separation of free ligands from bound ligands or immobilization of receptors and the scintillant on a solid-phase support. In this report, we describe the development of a homogeneous binding assay for a G protein-coupled receptor in the fluorescence polarization format. This homogeneous fluorescence polarization binding assay format is superior to the traditional binding methods because no radioisotope, separation step, or solid-phase support is required. The elimination of the separation step enhances detection of low-affinity ligands and enables a real-time, continuous readout of the binding activity in a high throughput 384-well microplate format.