Selection of Human Single Domain Antibodies (sdAb) Against Thymidine Kinase 1 and Their Incorporation Into sdAb-Fc Antibody Constructs For Potential Use In Cancer Therapy.

Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for liquid and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a tumor target. In order to examine the use of TK1 as a tumor target, it is necessary to develop therapeutics specific for TK1. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had a limit of detection of 3.9 ng/ml for 4-H-TK1_A1 and 1.9 ng/ml for 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mono nuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNC, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response by human MNCs against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 in tumor cells

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. Methods The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. Results Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. Conclusions The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

EnzyPha, an Engineered Helper Phage Developed to Overcome Most of the Limitations Regarding Filamentous Phage Titration and ELISA Tests

Background: Phage display method is a technology which enables expression of exogenous polypeptides on the surface of bacteriophage particles. Phage titration and ELISA are applied for measuring helper phage particles or polypeptide bearing phages and also evaluation of the interaction between polypeptide bearing phages and coated antigens, respectively. Although, several procedures have been introduced to perform phage titration and ELISA but they faced to some limitations such as being time consuming and expensive and also low reproducibility. Objective: We developed a new system called EnzyPha by engineering the M13KO7 expressing Secreted Acid Phosphatase of Mycobacterium tuberculosis (SapM enzyme) on its pIX protein for applying in colorimetric phage titration and ELISA methods. Methods: For this purpose, to evaluate the idea, colorimetric phage titration and ELISA were performed and compared to the traditional methods. Results: SapM enzyme was expressed on pIX protein of M13KO7 properly. The colorimetric phage titration and phage ELISA showed better and comparable results against the traditional approaches. Conclusion: The results showed that the proposed model would titrate phages more sensitive than plating titration method through a shorter timeframe. Moreover, it could be a better alternative to the routine phage ELISA due to time saving, cost effective and higher sensitivity.

Development of Real-Time Immuno-PCR Based on Phage Displayed an Anti-Idiotypic Nanobody for Quantitative Determination of Citrinin in Monascus

Citrinin (CIT) is a mycotoxin that has been detected in agricultural products, feedstuff, and Monascus products. At present, research has been performed to develop methods for CIT detection, mainly through TLC, HPLC, biosensor, and immunoassay. The immunoassay method is popular with researchers because of its speed, economy, simplicity, and ease of control. However, mycotoxins are inevitably introduced during the determination. Immunoassays require the use of toxins coupled to carrier proteins or enzymes to make competitive antigens. In this study, anti-idiotypic nanobody X27 as CIT mimetic antigen was used as non-toxic surrogate reagents in immunoassay. Therefore, the X27-based real-time immuno-PCR (rtIPCR) method had been established after optimal experiments of annealing temperature and amplification efficiency of real-time PCR, concentration of coating antibody, phage X27, and methyl alcohol. The IC50 value of the established method in the present study is 9.86 ± 2.52 ng/mL, which is nearly equivalent to the traditional phage ELISA method. However, the linear range is of 0.1–1000 ng/mL, which has been broadened 10-fold compared to the phage ELISA method. Besides, the X27-based rtIPCR method has no cross-reactivity to the common mycotoxins, like aflatoxin B1 (AFB1), deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). The method has also been applied to the determination of CIT in rice flour and flour samples, and the recovery was found to be in the range of 90.0–104.6% and 75.8–110.0% respectively. There was no significant difference in the results between the rtIPCR and UPLC–MS. The anti-idiotypic nanobody as a non-toxic surrogate of CIT makes rtIPCR a promising method for actual CIT analysis in Monascus products.

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

Application of Phage-displayed Single Chain Antibodies in Western Blot

A phage display single chain fragment variable library constructed on pIII protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.