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author:("Lou, tianlong")
1.  RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin 
Talanta  2013;117:273-280.
A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 minutes. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91% to 116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.
PMCID: PMC3909959  PMID: 24209341
RNA aptamer; botulinum neurotoxin; Surface Plasmon Resonance; aptasensor
2.  Impact of intrinsic affinity on functional binding and biological activity of EGFR antibodies 
Molecular cancer therapeutics  2012;11(7):1467-1476.
Aberrant expression and activation of epidermal growth factor receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280 fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from non-equilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.
PMCID: PMC4209485  PMID: 22564724
anti epidermal growth factor receptor antibody; affinity; avidity; phosphorylation; proliferation
3.  Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays 
Analytical biochemistry  2012;430(2):185-192.
Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples.
PMCID: PMC3589981  PMID: 22935296
Botulinum neurotoxin; Biodefense; ELISA; Protein microarray; Antibody; High-throughput assays
4.  Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors 
Cancer research  2011;71(6):2250-2259.
Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (MAb) which recognizes the same HER2 epitope (monovalent KDs ranging from 270nM to 0.56nM). Moderate affinity was associated with the highest tumor accumulation at 24hr and 120hr post i.v. injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity MAb were confined to the perivascular space of tumors with an average penetration of 20.4 +/− 7.5 microns from tumor blood vessels. Conversely, lowest affinity MAb exhibited a broader distribution pattern with an average penetration of 84.8 +/− 12.8 microns. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high affinity (trastuzumab) labeled with residualizing (111In-labeled) or non-residualizing (125I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Further, moderate to high affinity MAbs targeting the same HER2 epitope with monovalent affinity above 23nM had equal tumor accumulation of residualizing radiolabel over 120hrs. Results indicated equal tumor exposure, suggesting that MAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than rates of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.
PMCID: PMC3077882  PMID: 21406401
5.  Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies 
BMC Biochemistry  2011;12:58.
Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical.
In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.
In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.
PMCID: PMC3250939  PMID: 22085466
6.  Mouse and human lung fibroblasts regulate dendritic cell trafficking, airway inflammation, and fibrosis through integrin αvβ8–mediated activation of TGF-β 
The Journal of Clinical Investigation  2011;121(7):2863-2875.
The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models — conditional deletion in mouse fibroblasts of the TGF-β–activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts — we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.
PMCID: PMC3223836  PMID: 21646718
7.  A Single Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic Alpha-Exosite Binding Region 
Journal of molecular biology  2010;397(4):1106-1118.
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which while effective cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single domain VHH antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast displayed VHH with equilibrium dissociation constants (KD) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 by BoNT/A Lc. The most potent VHH (Aa1) had a solution KD for BoNT/A Lc of 1.47 × 10-10 M, an IC50 of 4.7 × 10-10 M, and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, the X-ray crystal structure of the BoNT/A Lc - Aa1 VHH complex was solved at 2.6 Å resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.
PMCID: PMC2903050  PMID: 20138889
botulinum neurotoxin type A; llama single VHH; single domain antibody; alpha-exosite; Naïve yeast-displayed library
8.  Specific Targeting of Proteolytic Activity for Tumor Detection in vivo 
Cancer research  2010;70(4):1505.
The cell surface protease membrane-type serine protease 1 [MT-SP1]/matriptase is often upregulated in epithelial cancers. A dysregulation in MT-SP1/matriptase levels with respect to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 [HAI-1] suggests that it is an increase in proteolytic activity that significantly differentiates malignant from normal tissue. Here we use antibodies to demonstrate that MT-SP1 is active on cancer cells and that this activity may be targeted for tumor detection in vivo. A proteolytic activity assay with the MT-SP1-positive human cancer cell lines MCF-7, HT29, LNCaP, and MDA-MB-468 showed that the antibodies, which inhibit recombinant catalytic MT-SP1, are able to bind and inhibit the full-length enzyme. The same experiment with the MT-SP1-negative breast cancer cell lines MDA-MB-231, COLO 320DM and HT1080 showed no inhibition of proteolysis. Fluorescent microscopy then confirmed localization of labeled antibodies to the surface of MT-SP1-positive cells. To evaluate these antibodies as probes for targeting MT-SP1 activity in vivo, 0.7-2 nanomoles of fluorescently labeled antibodies were administered to xenograft mouse cancer models. The antibodies localized to the MT-SP1-positive MCF-7 and MCF-7/Luc+ tumors (n=3), permitting visualization of MT-SP1 activity. Fluorescence was not observed in MT-SP1-negative MDA-MD-231/Luc+ tumors (n=2), suggesting that MT-SP1 activity is a novel biomarker for epithelial cancer and these antibodies provide a non-invasive method for detecting this activity in vivo.
PMCID: PMC2823079  PMID: 20145119
Cancer; MT-SP1; Matriptase; Protease Activity
9.  Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies 
Biosensors & bioelectronics  2009;25(1):179-184.
A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 hours was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.
PMCID: PMC2728038  PMID: 19643593
Quantum dot; immunoassay; botulinum toxin; fluorescence
10.  Extraction of BoNT/A, /B, /E, and /F with a Single, High Affinity Monoclonal Antibody for Detection of Botulinum Neurotoxin by Endopep-MS 
PLoS ONE  2010;5(8):e12237.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.
PMCID: PMC2923190  PMID: 20808925
11.  Antibody Protection against Botulinum Neurotoxin Intoxication in Mice▿  
Infection and Immunity  2009;77(10):4305-4313.
Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.
PMCID: PMC2747958  PMID: 19651864
12.  Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies 
PLoS ONE  2009;4(4):e5355.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.
PMCID: PMC2670495  PMID: 19399171

Results 1-12 (12)