Search results for: polyclonal antibody

Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

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Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity

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Authors: Sandra Tapin-Lingua, Katia Ruel, Jean-Paul Joseleau, Daouia Messaoudi, Olivier Fahy, Michel Petit-Conil

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The preservative efficacy of organic biocides is strongly related to their capacity of penetration and retention within wood tissues. The specific detection of the pyrethroid insecticide is currently obtained after extraction followed by chemical analysis by chromatography techniques. However visualizing the insecticide molecule within the wood structure requires specific probes together with microscopy techniques. Therefore, the aim of the present work was to apply a new methodology based on antibody-antigen recognition and electronic microscopy to visualize directly pyrethroids in the wood material. A polyclonal antibody directed against cypermethrin was developed and implement it on Pinus sylvestris wood samples coated with technical cypermethrin. The antibody was tested on impregnated wood and the specific recognition of the insecticide was visualized in transmission electron microscopy (TEM). The immunogold-TEM assay evidenced the capacity of the synthetic biocide to penetrate in the wood. The depth of penetration was measured on sections taken at increasing distances from the coated surface of the wood. Such results correlated with chemical analyzes carried out by GC-ECD after extraction. In addition, the immuno-TEM investigation allowed visualizing, for the first time at the ultrastructure scale of resolution, that cypermethrin was able to diffuse within the secondary wood cell walls.

Keywords: cypermethrin, insecticide, wood penetration, wood retention, immuno-transmission electron microscopy, polyclonal antibody

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Authors: Annisa Amalina, Lintang Sekar Sari, Khairunnisa Salsabila, Astya Gema Ramadhan, M. Fatkhi, Andani Eka Putra

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Gonorrhea is one of the sexually transmitted diseases by genito-genital, oro-genital and anogenital. Gonorrhea disease will cause complications if not treated properly. The diagnostic tool that has been used nowadays is microscopic. Thus a rapid diagnostic tool for gonorrhea is required, using polyclonal antibodies. The purpose of this study was to determine the effectiveness of injections of intravenous, subcutaneous and intracutaneous crude protein gonorrhea. The research method used in this research is experimental explorative. This research was conducted in Molecular Microbiology Laboratory of Faculty of Medicine, Andalas University for 3 months from April to June 2017. This study used 3 groups of rabbit with intravenous, subcutaneous, and intracutaneous injections. Each group was treated on days 1, 7, 21, and 28 with crude protein injection. After that, the examination of antibody levels held by using ELISA, followed by the antibody comparative tests contained in all three groups. The results examined by One Way ANOVA test on SPSS 21 and showed that there is no significant difference between intravenous, subcutaneous, and intracutaneous use p=0.69 (p < 0.05). However, there is an increased level (0.047 to 1.171) in antibodies from day 1 to day 14. In addition, subcutaneous use is preferred because it has minimal side effects compared to intravenous and intracutaneous use.

Keywords: crude protein, Neisseria gonorrhea, polyclonal antibodies, subcutaneous

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Authors: Parul Kulshreshtha, Subrata Sinha, Rakesh Bhatnagar

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This study was conducted by taking B. anthracis as a model pathogen. On infecting a host, B. anthracis secretes three proteins, namely, protective antigen (PA, 83kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These three proteins are the components of two anthrax toxins. PA binds to the cell surface receptors, namely, tumor endothelial marker (TEM) 8 and capillary morphogenesis protein (CMG) 2. TEM8 and CMG2 interact with LDL-receptor related protein (LRP) 6 for endocytosis of EF and LF. On entering the cell, EF acts as a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic adenosine monophosphate (cAMP). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MAPKK/MEK) close to their N-terminus. By secreting these two toxins, B.anthracis ascertains death of the host. Once the systemic levels of the toxins rise, antibiotics alone cannot save the host. Therefore, toxin-specific inhibitors have to be developed. In this wake, monoclonal antibodies have been developed for the neutralization of toxic effects of anthrax toxins. We created hybridomas by using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor of B. anthracis) to obtain anti-toxin antibodies. Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immunized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies from all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H8 and H10) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). The protective efficacy of H7, H8, H10 and H11 was investigated. H7, H8 and H10 were found to be protective. H11 was found to have disease enhancing characteristics in-vitro and in mouse model of challenge with B. anthracis. In this study the disease enhancing character of H11 monoclonal antibody and anti-rLFn polyclonal sera was investigated. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature both in-vitro and in-vivo. But combination of H11 with LETscFv (an scFv with VH and VL identical to H10 but lacking Fc region) could not abrogate the disease-enhancing character of H11 mAb. Therefore it was concluded that for suppression of disease enhancement, Fc portion was absolutely essential for interaction of H10 with H11. Our study indicates that the protective potential of an antibody depends equally on its idiotype/ antigen specificity and its isotype. A number of monoclonal and engineered antibodies are being explored as immunotherapeutics but it is absolutely essential to characterize each one for their individual and combined protective potential. Although new in the sphere of toxin-based diseases, it is extremely important to characterize the disease-enhancing nature of polyclonal as well as monoclonal antibodies. This is because several anti-viral therapeutics and vaccines have failed in the face of this phenomenon. The passive –immunotherapy thus needs to be well formulated to avoid any contraindications.

Keywords: immunotherapy, polyclonal, monoclonal, antibody-dependent disease enhancement

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Authors: Yemima Dani Riani, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of well known long-lived turtle. Its age can reach 100 years old. Senescence in green turtle is an interesting process to study because until now no clear explanation has been established about senescence at cellular or molecular level in this species. Since 1999, green turtle announced as an endangered species. Hence, establishment of fibroblast skin cell culture of green turtle may be material for future study of senescence. One common marker used for detecting senescence is telomere shortening. Reduced telomerase activity, the reverse transcriptase enzyme which adds TTAGGG DNA sequence to telomere end, may also cause senescence. The purpose of this research are establish and identify green turtle fibroblast skin cell culture and also compare telomere length and telomerase activity from passage 5 and 14. Primary cell culture made with primary explant method then cultured in Leibovitz-15 (Sigma) supplemented by 10% Fetal Bovine Serum (Sigma) and 100 U/mL Penicillin/Streptomycin (Sigma) at 30 ± 1oC. Cells identified with Rabbit Anti-Vimentin Polyclonal Antibody (Abcam) and Goat Polyclonal Antibody (Abcam) using confocal microscope (Zeiss LSM 170). Telomere length obtained using TeloTAGGG Telomere Length Assay (Roche) while telomerase activity obtained using TeloTAGGG Telomerase PCR ElisaPlus (Roche). Primary cell culture from green turtle skin had fibroblastic morphology and immunocytochemistry test with vimentin antibody proved the culture was fibroblast cell. Measurement of telomere length and telomerase activity showed that telomere length and telomerase activity of passage 14 was greater than passage 5. However, based on morphology, green turtle fibroblast skin cell culture showed senescent morphology. Based on the analysis of telomere length and telomerase activity, suspected fibroblast skin cell culture of green turtles is not undergo aging through telomere shortening.

Keywords: cell culture, chelonia mydas, telomerase, telomere, senescence

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Authors: Kieren Luellman, Makenzi Rockwell, Eduardo Callegari, Nichole Haag, Chun Wu

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Multiple sclerosis (MS) is an autoimmune disorder that damages the myelin sheath of neurons in the central nervous system. The presence of Acinetobacter bacteria and anti-Acinetobacter antibodies in MS patients has led to the hypothesis that the bacteria may contribute to MS pathogenesis. In this study, the protein sequences of Acinetobacter baumannii were compared to five peptides from three mammalian myelin proteins, i.e., Proteolipid Protein (PLP): PLP 139-151, PLP 178-191, Myelin Basic Protein (MBP): MBP 84-104 and Myelin Oligodendrocyte Glycoprotein (MOG): MOG 35-55 and MOG 92-106 respectively, known to induce experimental autoimmune encephalomyelitis (EAE), a condition similar to MS. We found 11 hits (i.e., with five or more amino acid sequence similarity) in Acinetobacter baumannii, which are identical or similar to PLP139-151, 32 hits to PLP178-191, 35 to MBP 84-104, 41 hits to MOG 35-55 and 26 hits to MOG92-106. In addition, Western blotting was used to assess possible interaction between the bacterial proteins and human anti-MBP, anti-MOG, and anti-PLP antibodies produced in rabbits, corresponding to MBP 84-104, MOG 35-55, and PLP 139-151, respectively. We found that both human Polyclonal anti-MOG antibody and anti-PLP antibody recognized a protein or more proteins of the same molecular mass of around 25 kDa. in Acinetobacter baumannii. The results suggested that this/these protein(s) might potentially serve as antigen(s) to induce anti-MOG antibody and anti-PLP antibody production in mammalian B cells. The proteomic study identified 433 hits, among which the sequence of Acinetobacter baumannii protein 491 subunit A matches a previously published enzyme Acinetobacter 3-Oxoadipate CoA-Transferase, in which a fragment of its peptide was observed to recognize MS patient serum via ELISA method. Our findings might pave the road to understanding one of the pathogenesis mechanisms of MS.

Keywords: multiple sclerosis, pathogenesis, Acinetobacter baumannii, antibody recognition

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Authors: Xiangjun Kong, Dongning Yao, Yuanjia Hu

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Therapeutic antibodies have become the most valuable and successful class of biopharmaceutical drugs, with a huge market potential and therapeutic advantages. Antibody patents are, accordingly, extremely important. As the technological limitation of the early stage of this field, the U. S. Patent and Trademark Offices (USPTO) have issued guidelines that suggest an exception for patents claiming a genus of antibodies that bind to a novel antigen, even in the absence of any experimental antibody production. This 'antibody exception' allowed for a broad scope on antibody claims, and led a global trend to patent antibodies without antibodies. Disputes around the pertinent patentability and written description issues remain particularly intense. Yet the validity of such patents had not been overtly challenged until Centocor v. Abbott, which restricted the broad scope of antibody patents and hit the brakes on the 'antibody exception'. The courts tend to uphold the requirement for adequate description of antibodies in the patent specifications, to avoid overreaching antibody claims. Patents following the 'antibody exception' are at risk of being found invalid for inadequately describing what they have claimed. However, the relation between the court and USPTO guidelines remains obscure, and the waning of the 'antibody exception' has led to further disputes around antibody patents. This uncertainty clearly affects patent applications, antibody innovations, and even relevant business performance. This study will give an overview of the emergence, debate, and waning usage of the 'antibody exception' in a number of enlightening cases, attempting to understand the specific concerns and the potential influence of antibody patents. We will then provide some possible strategies for antibody patenting, under the current considerations on the 'antibody exception'.

Keywords: antibody exception, antibody patent, USPTO (U. S. Patent and Trademark Offices) guidelines, written description requirement

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Authors: Shirin Jalili, Sadegh Hasannia, Hadi Shirzad, Afshin Khara

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Morphine is one of the most medicinally important analgesics and narcotics. Structurally, it is classified as an alkaloid because of the presence of nitrogen. Its structure is similar to that of codeine, thebaine, and heroin. An immunoassay to accurately discriminate between these analogous alkaloids would be highly beneficial. A key factor for such an assay is specificity with high sensitivity, which is totally dependent on the antibody employed. However, most antibodies against haptens are polyclonal serum antibodies that exhibit significant cross-reactivities with closely related compounds. The camel-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity, possessing unique properties compared to other conventional antibodies. In this study, a library containing the VHH genes of a camel immunized with with morphine conjugated BSA following phage display technology was generated. By screening the camel-derived variable region of the heavy chain cDNA phage display library with the ability to bind the desired hapten, we obtained some nanobodies that recognize this hapten. Phage display expression of the Nbs from this library and pannings against this hapten resulted in a clear enrichment of four distinct Nb-displaying phages with specificity for morphine that could be a potential target site for the development of new strategies for the development of a biosensor for detecting illicit drug.

Keywords: phage display, nanobody, Morphine-3, glucuronide, ELISA, biosensor

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Authors: Mudassar Hameed, Khushi Muhammad, Aamir Ghafoor, Masood Rabbani, Momena Habib, Jawad Nazir

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Comparative efficacy of three formulations (non-adjuvant, gel, and oil adjuvant) of monovalent and combined PPR and FMD virus vaccines was evaluated in goats. All kinds of monovalent PPRV vaccines elicited protective antibody titers at one-month post vaccination (PV) that remained so till six months PV. Monovalent non-adjuvant (NA) FMDV vaccine provoked non-protective antibody titers that declined to undetectable levels after three months. In case of combined vaccines, all of the formulations elicited protective antibody titers against PPRV in vaccinated animals which remained above that limit for six months. However, an exceptional immune response against FMDV was observed in combined NA vaccine group where antibody titers were extremely high and remained above protective level till 4 months PV in animals who received a single vaccine shot and till six months PV in booster group. Although, adjuvant or NA combined vaccines can induce protective antibody titers against both of the viruses within one month PV, but a booster vaccine shot is needed to retain protective antibody level for 6 months duration. Immune response elicited by combined vaccines is comparable or superior to the monovalent vaccines. Hence combined vaccine can be effectively used for the control and prevention of both of the diseases.

Keywords: antibody titer, protective, combined vaccine, non adjuvant

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Authors: Sonu Gandhi, Neena Capalash, Prince Sharma, C. Raman Suri

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A sensitive chemiluminescence immunoassay (CIA) for heroin and its major metabolites is reported. The method is based on the competitive reaction of horseradish peroxidase (HRP)-labeled anti-MAM antibody and free drug in spiked urine samples. A hapten-protein conjugate was synthesized by using acidic derivative of monoacetyl morphine (MAM) coupled to carrier protein BSA and was used as an immunogen for the generation of anti-MAM (monoacetyl morphine) antibody. A high titer of antibody (1:64,0000) was obtained and the relative affinity constant (Kaff) of antibody was 3.1×107 l/mol. Under the optimal conditions, linear range and reactivity for heroin, mono acetyl morphine (MAM), morphine and codeine were 0.08, 0.09, 0.095 and 0.092 ng/mL respectively. The developed chemiluminescence inhibition assay could detect heroin and its metabolites in standard and urine samples up to 0.01 ng/ml.

Keywords: heroin, metabolites, chemiluminescence immunoassay, horse radish peroxidase

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Authors: F. Kashanian, M. M. Masoudi, A. Akbari, A. Shamloo, M. R. Zand, S. S. Salehi

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Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this study, A-MNPs were synthesized via a simple precipitation reaction and directly immobilized Ep-CAM EBA-1 antibodies over superparamagnetic A-MNPs for Mucin BCA-225 in breast cancer cell. The samples were characterized by vibrating sample magnetometer (VSM), FT-IR spectroscopy, Tunneling Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These antibody-functionalized nontoxic A-MNPs were used to capture breast cancer cell. Through employing a strong permanent magnet, the magnetic separation was achieved within a few seconds. Antibody-Conjugated nontoxic Arginine-doped Fe₃O₄ nanoparticles have the potential for the future study to capture CTCs which are released from tumor tissue and for drug delivery, and these results demonstrate that the antibody-conjugated A-MNPs can be used in magnetic hyperthermia techniques for cancer treatment.

Keywords: tumor tissue, antibody, magnetic nanoparticle, CTCs capturing

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Authors: Seyed Mehrzad Pahlavani, Mozaffar Haji Jafari Anaraki, Sayma Mohammadi

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A total of 34700 day-old broilers were exposed to green, blue and yellow light using a light-emitting diode system for 6 weeks to investigate the effects of light wave length on antibody responses to Newcastle disease by HI test and the correlation with ELISA. 3 poultry house broiler farms with the same conditions was selected and the lightening system of each was set according to the requirement. Blood samples were taken from 20 chicks on days 1, 24 and 46 and the Newcastle virus specific antibody was titered in serum using HI an ELISA test. On day 24, the probability value of more than 0/05 was observed in HI and ELISA tests of all groups while at the end of breeding period, the average HI serum antibody titer was more in the green light than the yellow one while the blue light was not significantly different from both. At the last titration, the green light has got the highest titer of Newcastle antibodies. There were no significant differences of Newcastle antibody titers between all groups and ages in broiler pullets in ELISA. According to the sampling and analysis of HI and ELISA serum tests, there were no significant relationships between all broiler pullets breeding in green, blue and yellow light on days 24 and 46 and the P-value was more than 0/05. It is suggested that the monochromatic light is effective on broilers immunity against Newcastle disease.

Keywords: monochromatic light, Newcastle disease, HI test, ELISA test

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: Maryam Hashemi, Nematollah Razmi, Rasool Madani

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M. paratuberculosis is a slow growing mycobactin dependent mycobacterial species known to be the causative agent of Johne’s disease in all species of domestic ruminants worldwide. JD is characterized by gradual weight loss; decreased milk production. Excretion of the organism may occur for prolonged periods (1 to 2.5 years) before the onset of clinical disease. In recent years, researchers focus on identification a specific antigen of MAP to use in diagnosis test and preparation of effective vaccine. In this paper, for production of polyclonal antibody against proteins of Mycobacterium avium paratuberculosis cell wall a rabbit immunization at a certain time period with antigen. After immunization of the animal, blood samples were collected from the rabbit for producing enriched serum. Antibodies were purified with ion exchange chromatography. For exact measurement of interaction, western blotting test was used and as it is demonstrated in the study, sharp bands appear in nitrocellulose paper and specific bands were 50 and 150 KD molecular weight. These were indicating immunodominant proteins.

Keywords: immunodominant, paratuberculosis, Western blotting, cell wall proteins, protein purification

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Authors: Camilla Trevor, Matthew K. Higgins, Andrea Gonzalez-Munoz, Mark Carrington

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Trypanosomiasis is a devastating disease affecting both humans and livestock in sub-Saharan Africa. The diseases are caused by infection with African trypanosomes, protozoa transmitted by tsetse flies. Treatment currently relies on the use of chemotherapeutics with ghastly side effects. Here, we describe the development of effective antibody-drug conjugates that target the T. brucei transferrin receptor. The receptor is essential for trypanosome growth in a mammalian host but there are approximately 12 variants of the transferrin receptor in the genome. Two of the most divergent variants were used to generate recombinant monoclonal immunoglobulin G using phage display and we identified cross-reactive antibodies that bind both variants using phage ELISA, fluorescence resonance energy transfer assays and surface plasmon resonance. Fluorescent antibodies were used to demonstrate uptake into trypanosomes in culture. Toxin-conjugated antibodies were effective at killing trypanosomes at sub-nanomolar concentrations. The approach of using antibody-drug conjugates has proven highly effective.

Keywords: antibody-drug conjugates, phage display, transferrin receptor, trypanosomes

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: Yasser A. Ahmed, Juleen M Dickson, Evan S. Krystofiak, Julie A. Oliver

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Cancer cells urgently need folate to support their rapid division. Folate receptors (FR) are over-expressed on a wide range of tumor cells, including breast cancer cells. FR are distributed over the entire surface of cancer cells, but are polarized to the apical surface of normal cells. Targeting of cancer cells using specific surface molecules such as folate receptors may be one of the strategies used to kill cancer cells without hurting the neighing normal cells. The aim of the current study was to try a method of SEM detecting FR in a murine breast cancer cell model (4T1 cells) using secondary antibody conjugated to gold or gold-coated magnetite nanoparticles. 4T1 cells were suspended in RPMI medium witth FR antibody and incubated with secondary antibody for fluorescence microscopy. The cells were cultured on 30mm Thermanox coverslips for 18 hours, labeled with FR antibody then incubated with secondary antibody conjugated to gold or gold-coated magnetite nanoparticles and processed to scanning electron microscopy (SEM) analysis. The fluorescence microscopy study showed strong punctate FR expression on 4T1 cell membrane. With SEM, the labeling with gold or gold-coated magnetite conjugates showed a similar pattern. Specific labeling occurred in nanoparticle clusters, which are clearly visualized in backscattered electron images. The 4T1 tumor cell model may be useful for the development of FR-targeted tumor therapy using gold-coated magnetite nano-particles.

Keywords: cancer cell, nanoparticles, cell culture, SEM

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Authors: Kolja Them, Priyal Chikhaliwala, Sudeshna Chandra

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Purpose: The purpose of this work is to examine possibilities for noninvasive imaging and identification of tumor markers for cancer diagnosis. The proposed method uses antibody conjugated iron oxide nanoparticles and multicolor Magnetic Particle Imaging (mMPI). The method has the potential for radiation exposure free real-time estimation of local tumor marker concentrations in vivo. In this study, the method is applied to human Alpha-1-Fetoprotein. Materials and Methods: As tracer material AFP antibody-conjugated Dendrimer-Fe3O4 nanoparticles were used. The nanoparticle bioconjugates were then incubated with bovine serum albumin (BSA) to block any possible nonspecific binding sites. Parts of the resulting solution were then incubated with AFP antigen. MPI measurements were done using the preclinical MPI scanner (Bruker Biospin MRI GmbH) and the multicolor method was used for image reconstruction. Results: In multicolor MPI images the nanoparticles incubated only with BSA were clearly distinguished from nanoparticles incubated with BSA and AFP antigens. Conclusion: Tomographic imaging of human tumor marker Alpha-1-Fetoprotein is possible using AFP antibody conjugated iron oxide nanoparticles in presence of BSA. This opens interesting perspectives for cancer diagnosis.

Keywords: noninvasive imaging, tumor antigens, antibody conjugated iron oxide nanoparticles, multicolor magnetic particle imaging, cancer diagnosis

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Authors: Xia Huo

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Children are more vulnerable to environmental toxicants compared to adults, and their developing immune system is among the most sensitive targets regarding toxicity of environmental toxicants. Studies have found that exposure to environmental toxicants is associated with impaired immune function in children, but only a few studies have focused on the relationship between environmental toxicant exposure and vaccine antibody potency and immunoglobulin (Ig) levels in children. These studies investigated the associations of exposure to polychlorinated biphenyls (PCBs), perfluorinated compounds (PFCs), heavy metals (Pb, Cd, As, Hg) and PM2.5 with the serum-specific antibody concentrations and Ig levels against different vaccines, such as anti-Hib, tetanus, diphtheria toxoid, and analyze the possible mechanisms underlying exposure-related alterations of antibody titers and Ig levels against different vaccines. Results suggest that exposure to these toxicants is generally associated with decreased potency of antibodies produced from childhood immunizations and an overall deficiency in the protection the vaccines provide. Toxicant exposure is associated with vaccination failure and decreased antibody titers, and increased risk of immune-related diseases in children by altering specific immunoglobulin levels. Age, sex, nutritional status, and co-exposure may influence the effects of toxicants on the immune function in children. Epidemiological evidence suggests that exposure-induced changes to humoral immunerelated tissue/cells/molecules response to vaccines may have predominant roles in the inverse associations between antibody responsiveness to vaccines and environmental toxicants. These results help us to conduct better immunization policies for children under environmental toxicant burden.

Keywords: environmental toxicants, immunotoxicity, vaccination, antibodies, children's health

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Authors: Teodora Mocan, Lucian Mocan, Cornel Iancu, Flaviu A. Tabaran, Bartos Dana, Matea Cristian

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Antimicrobial resistance, which threatens the efficacy of the existing antibiotics represents a worldwide public health issue. At the current time, vancomycin is the only responsive treatment although has significant cytotoxicity, is partially effective and it is poorly retained by infected tissues. From a clinical point of view, attractive alternative approaches for treating such Meticillin-Resistant Staphylococcus Aureus (MRSA) strains would be using agents that cause physical damage to the bacteria. Modular nanopharmaceuticals systems are being designed to address all of these multifunctional capabilities for the ideal bacterial treatment, with the ability to mix and match appropriate functions. Here we present a novel method of selective laser photothermal ablation of MRSA bacteria mediated by gold nanoparticles bound to PBP antibody against PBP protein located on the MRSA surface.

Keywords: MRSA, laser, nanoparticle, antibody

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Authors: F. Motamedi Sedeh, Sh. Chahardoli, H. Mahravani, N. Harzandi, M. Sotoodeh, S. K. Shafaei

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Foot-and-mouth disease virus (FMDV) is the most economically important disease of livestock. The aim of the study is inactivation of FMD virus type O/IRN/2010 by electron beam without antigenic changes as electron radio vaccine. The BALB/C mice were divided into three groups, each group containing five mice. Three groups of mice were inoculated with conventional vaccine and electron beam irradiated vaccine FMDV type O/IRN/2010 subcutaneously three weeks interval, the final group as negative control. The sera were separated from the blood samples of mice 14 days after last vaccination and tested for the presence of antibodies against FMDV type O/IRN/2010 by serum neutralization test. The Serum Neutralization Test (SNT) was carried out and antibody titration was calculated according to the Kraber protocol. The results of the SNT in three groups of mice showed the titration of neutralizing antibody in the vaccinated mice groups; electron radio vaccine and conventional vaccine were significantly higher than negative control group (P<0.05). Therefore, the radio vaccine is a good candidate to immunize animals against FMDV type O/IRN/2010.

Keywords: FMDV type O/IRN/2010, neutralizing antibody response, electron beam, radio vaccine

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Authors: Faris Mohsin Alabeedi

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Pemphigus Vulgaris (PV) is a life-threatening autoimmune blistering disease characterized by the presence of autoantibodies directed against the epidermis's surface proteins. There are two forms of PV, mucocutaneous and mucosal-dominant PV. Disruption of the cell junctions is a hallmark of PV due to the autoantibodies targeting the desmosomal cadherins, desmoglein-3 (Dsg3) and desmoglein-1, leading to acantholysis in the skin and mucous membrane. Although the pathogenesis of PV is known, the detailed molecular events remain not fully understood. Our recent study has shown that both the PV sera and pathogenic anti-Dsg3 antibody AK23 can induce ROS and cause oxidative stress in cultured keratinocytes. In line with our finding, other independent studies also demonstrate oxidative stress in PV. Since Nrf2 plays a crucial role in cellular anti-oxidative stress response, we hypothesize that the expression of Nrf2 may alter in PV. Thus, treatment of cells with PV sera or AK23 may cause changes in Nrf2 expression and distribution. The purpose of this study was to examine the effect of AK23 and PV sera on Nrf2 in a normal human keratinocyte cell line, such as NTERT cells. Both a time-course and dose-dependent experiments with AK23, alongside the matched isotype control IgG, were performed in keratinocyte cultures and analysed by immunofluorescence for Nrf2 and Dsg3. Additionally, the same approach was conducted with the sera from PV patients and healthy individuals that served as a control in this study. All the fluorescent images were analysed using ImageJ software. Each experiment was repeated twice. In general, variations were observed throughout this study. In the dose-response experiments, although enhanced Dsg3 expression was consistently detected in AK23 treated cells, the expression of Nrf2 showed no consistent findings between the experiments, although changes in its expression were noticeable in cells treated with AK23. In the time-course study, a trend with induction of Nrf2 over time was shown in control cells treated with mouse isotype IgG. Treatment with AK23 showed a reduction of Nrf2 in a time-dependent manner, especially at the 24-hour time point. However, the earlier time points, such as 2 hours and 6 hours with AK23 treatments, detected somewhat variations. Finally, PV sera caused a decrease of Dsg3, but on the other hand, variations were observed in Nrf2 expression in PV sera treated cells. In general, PV sera seemed to cause a reduction of Nrf2 in the majority of PV sera treated samples. In addition, more pronounced cytoplasmic expression of Nrf2 has been observed in PV sera treated cells than those treated with AK23, suggesting that polyclonal and monoclonal IgG might induce a different effect on Nrf2 expression and distribution. Further experimental studies are crucial to obtain a more coincide global view of Nrf2-mediated gene regulation. In particular, Pemphigus Voulgaris studies assessing how the Nrf2-dependent network changes from a physiological to a pathological condition can provide insight into disease mechanisms and perhaps initiate further treatment approaches.

Keywords: pemphigus vulgaris, monoclonal antibody against desmoglein-3, Nrf2 oxidative stress, keratinocyte cultures

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Authors: Ibrahim Aly, Manal Ahmed, Mahmoud M. El-Shall

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Filariasis is a parasitic disease caused by small roundworms. The filarial worms are transmitted and spread by blood-feeding black flies and mosquitoes. Lymphatic filariasis (Elephantiasis) is caused by Wuchereriabancrofti, Brugiamalayi, and Brugiatimori. Elimination of Lymphatic filariasis necessitates an increasing demand for valid, reliable, and rapid diagnostic kits. Nanodiagnostics involve the use of nanotechnology in clinical diagnosis to meet the demands for increased sensitivity, specificity, and early detection in less time. The aim of this study was to evaluate the nano-based enzymelinked immunosorbent assay (ELISA) and novel nano-chip card as a rapid test for detection of filarial antigen in serum samples of human filariasis in comparison with traditional -ELISA. Serum samples were collected from an infected human with filarial gathered across Egypt's governorates. After receiving informed consenta total of 45 blood samples of infected individuals residing in different villages in Gharbea governorate, which isa nonendemic region for bancroftianfilariasis, healthy persons living in nonendemic locations (20 persons), as well as sera from 20 other parasites, affected patients were collected. The microfilaria was checked in thick smears of 20 µl night blood samples collected during 20-22 hrs. All of these individuals underwent the following procedures: history taking, clinical examination, and laboratory investigations, which included examination of blood samples for microfilaria using thick blood film and serological tests for detection of the circulating filarial antigen using polyclonal antibody- ELISA, nano-based ELISA, and nano-chip card. In the present study, a recently reported polyoclonal antibody specific to tegumental filarial antigen was used in developing nano-chip card and nano-ELISA compared to traditional ELISA for the detection of circulating filarial antigen in sera of patients with bancroftianfilariasis. The performance of the ELISA was evaluated using 45 serum samples. The ELISA was positive with sera from microfilaremicbancroftianfilariasis patients (n = 36) with a sensitivity of 80 %. Circulating filarial antigen was detected in 39/45 patients who were positive for circulating filarial antigen using nano-ELISA with a sensitivity of 86.6 %. On the other hand, 42 out of 45 patients were positive for circulating filarial antigen using nano-chip card with a sensitivity of 93.3%.In conclusion, using a novel nano-chip assay could potentially be a promising alternative antigen detection test for bancroftianfilariasis.

Keywords: lymphatic filariasis, nanotechnology, rapid diagnosis, elisa technique

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Authors: M. M. Rahman, A. Liu, A. Frostegard, J. Frostegard

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The human immune system plays an essential role in cardiovascular disease (CVD) and atherosclerosis. Our earlier studies showed that major immunocompetent cells including T cells are activated by phosphorylcholine epitope. Further, we have determined for the first time in a clinical cohort that antibodies against phosphorylcholine (anti-PC) are negatively and independently associated with the development of atherosclerosis and thus a low risk of cardiovascular diseases. It is still unknown whether activated T cells play a role in anti-PC production. Here we aim to clarify the role of T cells in anti-PC production. B cell alone, or with CD3 T, CD4 T or with CD8 T cells were cultured in polystyrene plates to examine anti-PC IgM production. In addition to mixed B cell with CD3 T cell culture, B cells with CD3 T cells were also cultured in transwell co-culture plates. Further, B cells alone and mixed B cell with CD3 T cell cultures with or without anti-HLA 2 antibody were cultured for 6 days. Anti-PC IgM was detected by ELISA in independent experiments. More than 8 fold higher levels of anti-PC IgM were detected by ELISA in mixed B cell with CD3 T cell cultures in comparison to B cells alone. After the co-culture of B and CD3 T cells in transwell plates, there were no increased antibody levels indicating that B and T cells need to interact to augment anti-PC IgM production. Furthermore, anti-PC IgM was abolished by anti-HLA 2 blocking antibody in mixed B and CD3 T cells culture. In addition, the lack of increased anti-PC IgM in mixed B with CD8 T cells culture and the increased levels of anti-PC in mixed B with CD4 T cells culture support the role of helper T cell for the anti-PC IgM production. Atherosclerosis is a major cause of cardiovascular diseases, but anti-PC IgM is a protection marker for atherosclerosis development. Understanding the mechanism involved in the anti-PC IgM regulation could play an important role in strategies to raise anti-PC IgM. Studies suggest that anti-PC is T-cell independent antibody, but our study shows the major role of T cell in anti-PC IgM production. Activation of helper T cells by immunization could be a possible mechanism for raising anti-PC levels.

Keywords: anti-PC, atherosclerosis, aardiovascular diseases, phosphorylcholine

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Authors: Robert van Ling, Alexander Schwahn, Shanhua Lin, Ken Cook, Frank Steiner, Rowan Moore, Mauro de Pra

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Purpose: Demonstration of fast high resolution charge variant analysis for monoclonal antibody (mAb) therapeutics within 5 minutes. Methods: Three commercially available mAbs were used for all experiments. The charge variants of therapeutic mAbs (Bevacizumab, Cetuximab, Infliximab, and Trastuzumab) are analyzed on a strong cation exchange column with a linear pH gradient separation method. The linear gradient from pH 5.6 to pH 10.2 is generated over time by running a linear pump gradient from 100% Thermo Scientific™ CX-1 pH Gradient Buffer A (pH 5.6) to 100% CX-1 pH Gradient Buffer B (pH 10.2), using the Thermo Scientific™ Vanquish™ UHPLC system. Results: The pH gradient method is generally applicable to monoclonal antibody charge variant analysis. In conjunction with state-of-the-art column and UHPLC technology, ultra fast high-resolution separations are consistently achieved in under 5 minutes for all mAbs analyzed. Conclusion: The linear pH gradient method is a platform method for mAb charge variant analysis. The linear pH gradient method can be easily optimized to improve separations and shorten cycle times. Ultra-fast charge variant separation is facilitated with UHPLC that complements, and in some instances outperforms CE approaches in terms of both resolution and throughput.

Keywords: charge variants, ion exchange chromatography, monoclonal antibody, UHPLC

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Authors: Mombaur Busisiwe, Lesosky Maia, Liebenberg Lisa, Heckmann Jeannine

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Introduction: To assess age- and gender-specific incidence rates (IR) of acetylcholine receptor (AChR)-antibody positive myasthenia gravis (MG) in South Africa, and geographical variation in incidence. Methods: IRs were calculated from positive AChR antibody laboratory data between 2011 and 2012, using 2011 population census data. Results:890 individuals were seropositive, for an annual IR of 8.5 per million. Age-standardized IR for early- (< 50) and late-onset (≥ 50) MG were 4.1 and 24 per million, respectively, and for juveniles, 4.3 per million. The IR between provinces ranged from 1 to 19 per million. Conclusions: In this Southern hemisphere African population, the overall IR and peak IR (in older men) for seropositive MG is comparable to that in Europe and North America, arguing against environmental factors. However, IRs may be higher among children with African genetic ancestry. Geographical variation in incidence underscores the importance of outreach programs for regions with limited resources.

Keywords: incidence rates (IR), acetylcholine receptor (AChR), myasthenia gravis (MG), South Africa

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Authors: Teklay Gebrecherkos

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Background: Serological testing for SARS-CoV-2 plays an important role in epidemiological studies, in aiding the diagnosis of COVID-19 and assess vaccine responses. Little is known about the dynamics of SARS-CoV-2 serology in African settings. Here, we aimed to characterize the longitudinal antibody response profile to SARS-CoV-2 in Ethiopia. Methods: In this prospective study, a total of 102 PCR-confirmed COVID-19 patients were enrolled. We obtained 802 plasma samples collected serially. SARS-CoV-2 antibodies were determined using four lateral flow immune assays (LFIAs) and an electrochemiluminescent immunoassay. We determined longitudinal antibody response to SARS-CoV-2 as well as seroconversion dynamics. Results: Serological positivity rate ranged between 12%-91%, depending on timing after symptom onset. There was no difference in the positivity rate between severe and non-severe COVID-19 cases. The specificity ranged between 90%-97%. Agreement between different assays ranged between 84%-92%. The estimated positive predictive value (PPV) for IgM or IgG in a scenario with seroprevalence at 5% varies from 33% to 58%. Nonetheless, when the population seroprevalence increases to 25% and 50%, there is a corresponding increase in the estimated PPVs. The estimated negative-predictive value (NPV) in a low seroprevalence scenario (5%) is high (>99%). However, the estimated NPV in a high seroprevalence scenario (50%) for IgM or IgG is reduced significantly from 80% to 85%. Overall, 28/102 (27.5%) seroconverted by one or more assays tested within a median time of 11 (IQR: 9–15) days post symptom onset. The median seroconversion time among symptomatic cases tended to be shorter when compared to asymptomatic patients [9 (IQR: 6–11) vs. 15 (IQR: 13–21) days; p = 0.002]. Overall, seroconversion reached 100% 5.5 weeks after the onset of symptoms. Notably, of the remaining 74 COVID-19 patients included in the cohort, 64 (62.8%) were positive for antibodies at the time of enrollment, and 10 (9.8%) patients failed to mount a detectable antibody response by any of the assays tested during follow-up. Conclusions: Longitudinal assessment of antibody response in African COVID-19 patients revealed heterogeneous responses. This underscores the need for a comprehensive evaluation of serum assays before implementation. Factors associated with failure to seroconvert need further research.

Keywords: COVID-19, antibody, rapid diagnostic tests, ethiopia

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Authors: Abu Salim Mustafa

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The comparisons of mycobacterial genomes have identified several Mycobacterium tuberculosis-specific genomic regions that are absent in other mycobacteria and are known as regions of differences. Due to M. tuberculosis-specificity, the peptides encoded by these regions could be useful in the specific diagnosis of tuberculosis. To explore this possibility, overlapping synthetic peptides corresponding to 39 proteins predicted to be encoded by genes present in regions of differences were tested for antibody-reactivity with sera from tuberculosis patients and healthy subjects. The results identified four immunodominant peptides corresponding to four different proteins, with three of the peptides showing significantly stronger antibody reactivity and rate of positivity with sera from tuberculosis patients than healthy subjects. The fourth peptide was recognized equally well by the sera of tuberculosis patients as well as healthy subjects. Predication of antibody epitopes by bioinformatics analyses using ABCpred server predicted multiple linear epitopes in each peptide. Furthermore, peptide sequence analysis for sequence identity using BLAST suggested M. tuberculosis-specificity for the three peptides that had preferential reactivity with sera from tuberculosis patients, but the peptide with equal reactivity with sera of TB patients and healthy subjects showed significant identity with sequences present in nob-tuberculous mycobacteria. The three identified M. tuberculosis-specific immunodominant peptides may be useful in the serological diagnosis of tuberculosis.

Keywords: genomic regions of differences, Mycobacterium tuberculossis, peptides, serodiagnosis

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Authors: Emad Al-Abdallat, Faris G. Bakri, Azmi Mahafza, Rayyan Al Ali, Nidaa Ababneh, Ahmed Idhair

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Background: Morgues are high-risk areas for the spread of infection from the cadavers to the staff during the postmortem examination. Infection can spread from corpses to workers by the airborne route, by direct contact, or from needle and sharp object injuries. Objective: Knowledge about the prevalence of these infections among autopsies is prudent to appreciate any risk of transmission and to further enforce safety measures. Method: A total of 242 autopsies were tested. Age ranged from 3 days to 94 years (median 75.5 years, mean 45.3 (21.9 ± SD)). There were 172 (71%) males. Results: The cause of death was considered natural in 137 (56.6%) cases, accidental in 89 (36.8%), homicidal in 9 (3.7%), suicidal in 4 (1.7%), and unknown in 3 (1.2%). Hepatitis B surface antigen was positive in 5 (2.1%) cases. Hepatitis C virus antibody was detected in 5 (2.1%) cases and the hepatitis C virus polymerase chain reaction was positive in 2 of them (0.8%). HIV antibody was not detected in any of the cases. Conclusions: Autopsies can be associated with exposure to blood borne viruses. Autopsies performed during the study period were tested for hepatitis B surface antigen, hepatitis C virus antibody, and human immunodeficiency virus antibody. Positive tests were subsequently confirmed by polymerase chain reaction. There is low prevalence of infections with these viruses in our autopsy cases. However, the risk of transmission remains a threat. Healthcare workers in the forensic departments should adhere to standard precautions.

Keywords: autopsy, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Jordan

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Authors: Mohammad Javad Mehrabanpour, Mohammad Hosein Hosseini, Ali Shirazi, Dorsa Mehrabanpour

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Respiratory infections are the most important diseases that affect poultry. Ornithobacterium rhinotracheale is a bacterium that causes respiratory infections including alveolar inflation and pneumonia in birds. The aim of this study was to evaluated antibody titer against Ornitobacterium rhinotracheal in layer chicken sera after vaccination with an experimental ORT vaccine that produced in Razi Vaccine and Serum Research Institute. Cultured bacteria were inactivated by formalin, and controlled tests were conducted on it. The obtained antigens were formulated using Montanide oil and were homogenized using homogenizer. Eighty SPF chickens were kept until the age of 14 days under existing standards for temperature, humidity, and light. At the age of 14 days, chickens were divided into 3 groups. The first group included 50 chickens injected with prepared ORT vaccine, the second group, as control group, included 15 chickens injected with sterile PBS to get stress of infection and the third group included 15 chickens with no injection performed to them. All 3 groups were kept in separate cages at same room. Blood samples were regularly taken from the chickens every week for serum separation and evaluation of antibody titer. During the fifth week post vaccination, booster vaccine was injected into the chickens of vaccinated group. The chickens were inspected every day in terms of mortality as well as any injection site reactions. Three weeks after the booster injection, blood samples were taken from all chickens of all groups, and sera were isolated. The sera of immunized (vaccinated) SPF chickens with ORT vaccine as well as that of SPF chickens in the control groups were reviewed according to the recommendations of ELISA kit manufacturer to examine the chicken’s humeral immune response to the studied vaccine. Potency, stability and sterility tests were also performed on the above mentioned vaccine. Results obtained indicate high antibody titer in sera of chickens vaccinated with experimental ORT vaccine as compared with the control groups that emphasize the ability of experimentally prepared ORT vaccine to stimulate humoral immune response of chicken. After the second injection, antibody titer increased and remained almost stable up to 9 weeks after the injection. ORT vaccine can cause potency in chickens and can protect them against disease.

Keywords: antibody, layer chicken, Ornithobactrium rhinotracitis, vaccine

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