Human Antibodies - Volume 26, issue 4

Authors: Valedkarimi, Zahra | Nasiri, Hadi | Aghebati-Maleki, Leili | Abdolalizadeh, Jalal | Esparvarinha, Mojghan | Majidi, Jafar

Article Type: Research Article

Abstract: In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab’)2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, …two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab’)2. Non-reduced SDS-PAGE for determining the purity of F(ab’)2 fragment resulted in one band in 100 kDa corresponds to F(ab’)2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab’)2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab’)2 fragment worked as efficiently as the commercial one. Show more

Keywords: Polyclonal antibody, F(ab’)2 fragment, FITC conjugation, immunization, enzyme digestion

DOI: 10.3233/HAB-180336

Citation: Human Antibodies, vol. 26, no. 4, pp. 171-176, 2018

Authors: Wezler, Xenia | Dübel, Stefan | Schirrmann, Thomas

Article Type: Research Article

Abstract: ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar …excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity. Show more

Keywords: ImmunoRNase, human RNase A superfamily, RNases 1 to 8, human antibody, CD30, lymphoma, antibody-based therapy

DOI: 10.3233/HAB-180337

Citation: Human Antibodies, vol. 26, no. 4, pp. 177-192, 2018

Authors: Willis, R. | Heslop, O. | Bodonaik, N. | Thame, M. | Kumar, U. | Aung, M. | Kellier, A. | Drysdale, S. | Leon, V. | Smikle, M.

Article Type: Research Article

Abstract: BACKGROUND: Pneumococcal serotypes circulating in any population vary over time and between countries and impacts the effectiveness of pneumococcal vaccination. OBJECTIVE: This study investigated the epidemiology of pneumococcal disease in Jamaica. METHODS: Streptococcus pneumoniae isolates (n = 349) along with demographic and clinical information were collected from patients presenting at the 4 major hospitals in Jamaica over a 2-year period. Serotyping was done using latex agglutination tests and the Quellung reaction assay. RESULTS: Invasive pneumococcal disease (IPD) incidence was 45.4/100,000 in children under 5 yrs and 16.3/100,000 …in adults over 65 yrs. Thirteen serogroups were identified among the 120 isolates subjected to grouping; the most common being serogroups: 19 (22/120,18.3%), 6 (20/120,16.7%), 14 (20/120,16.7%), 23 (18/120,15.0%), 3 (11/120,9.2%) and nontypeable (8/120,2.3%). The estimated vaccine coverage rates for the PCV7 and PCV13 vaccines in children less than 5 yrs were 82.5% and 88.7% respectively. The 23-valent PPV23 provided 100% coverage rate in adults over 65 yrs and 82.9% coverage rate for the entire population. CONCLUSIONS: Pneumococcal vaccine coverage rates in Jamaica are comparable to those reported in certain developed countries and higher than in other developing countries. The high incidence of IPD in the paediatric population indicates that routine vaccination would be beneficial. Show more

Keywords: Streptococcus pneumoniae, pneumococcus, vaccine, serotype, Serogroup, Jamaica, Epidemiology

DOI: 10.3233/HAB-180338

Citation: Human Antibodies, vol. 26, no. 4, pp. 193-199, 2018

Authors: Moravvej, Hamideh | Tabatabaei-Panah, Pardis-Sadat | Ebrahimi, Elaheh | Esmaeili, Nafiseh | Ghaderian, Sayyed Mohammad Hossein | Ludwig, Ralf J. | Akbarzadeh, Reza

Article Type: Research Article

Abstract: BACKGROUND: TNF-α -308G/A polymorphism has been investigated in few studies for an association with susceptibility to bullous pemphigoid (BP) and alopecia areata (AA). Yet, these findings had so far not been independently replicated, and no data on a possible association of TNFα -308G/A polymorphism with these diseases in Iranian population were available. OBJECTIVES: In the present study, a possible effect of TNF-α -308G/A variation on susceptibility to BP or AA disease was evaluated. METHODS: Genomic DNA was extracted from the blood of the patients …with BP and AA as well as control subjects which genotyped for the TNF-α -308 G/A polymorphism. TNF-α gene expression levels were analyzed by real-time RT-PCR. RESULTS: No association was observed between the TNF-α -308 G/A variation and susceptibility to BP or AA diseases in our Iranian cohort. In contrast to AA patients, expression of TNF-α gene was significantly higher in BP patients compared to control group. TNF-α gene was found to be similarly expressed in mutant and wild-type genotypes. CONCLUSIONS: TNF-α -308G/A polymorphism is not associated with the risk to develop of BP and AA in our Iranian cohort. Furthermore, this polymorphism is contributed to altering the levels of gene expression in BP disease. Show more

Keywords: Tumor necrosis factor-α, polymorphism, gene expression, bullous pemphigoid, alopecia areata

DOI: 10.3233/HAB-180339

Citation: Human Antibodies, vol. 26, no. 4, pp. 201-207, 2018

Authors: Hemati, Atefeh | Azarnia, Mahnaz | Hossein Modarressi, Mohammad | Rahimi, Amir

Article Type: Research Article

Abstract: BACKGROUND: Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. OBJECTIVE: Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. METHODS: To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular …laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. RESULTS: By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. CONCLUSIONS: So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases. Show more

Keywords: Hybridoma, monoclonal antibodies, spermatogenesis, testis, fertilization

DOI: 10.3233/HAB-180340

Citation: Human Antibodies, vol. 26, no. 4, pp. 209-218, 2018

Authors: Sayad, Arezou | Salmani, Tayyebali | Hemmesi, Maryam Kazemzadeh | Ganji, Maziar | Ghafouri-Fard, Soudeh | Hatami, Mahsa | Soudyab, Mohammad | Taheri, Mohammad

Article Type: Research Article

Abstract: Multiple sclerosis (MS) is an autoimmune disease characterized by recurrent episodes of demyelination and loss of oligodendrocytes. The demyelination process is caused by various subsets of CD4+ T cells with a Th1 and Th17 phenotype. The retinoid acid-related orphan receptor A (RORA ) is expressed in Th17 cells and promote Th17 differentiation. In this study, we compared the expression level of RORA gene in the blood of 50 relapsing-remitting MS (RRMS) patients who were treated with IFN-β and 50 healthy controls by TaqMan Quantitative Real-Time PCR. We found that RORA expression …was significantly down-regulated in MS patients compared with controls (P = 0.006). However, there was no significant correlation between RORA gene expression and Kurtzke Expanded Disability Status Scale (EDSS). Our findings suggest a possible contribution of IFN-β in the downregulation of RORA . In addition, RORA downregulation may be a potential indicator of positive response to interferon beta treatment of multiple sclerosis patients. Show more

Keywords: Multiple sclerosis, RORA, IFN-β

DOI: 10.3233/HAB-180341

Citation: Human Antibodies, vol. 26, no. 4, pp. 219-224, 2018

Authors: Zarei, Adi Essam | Redwan, Elrashdy M.

Article Type: Research Article

Abstract: In this national comprehensive seroprevalence study, indirect ELISA test was used to evaluate Haemophilus influenzae type b (Hib) anti-polyribosyl-ribitol phosphate (PRP) total antibodies (IgM, IgG, IgA) in 1,003 sera samples from routine medical check-up of healthy individuals attending the local medical facility in Jeddah, Saudi Arabia in the period from February 2014 to January 2016. Serum anti-CPS antibodies confer immunity against invasive Hib disease. An anti-CPS concentration of ⩾  0.15 μ g/mL is believed to be a serological indication for short-term immunity protection against invasive Hib disease, while a concentration of ⩾ …1.0 μ g/mL is believed to be long-term protective. Results showed higher level of anti-Hib IgG (2.41 μ g/ml average geometric mean concentration (GMC) regardless of age and gender, followed by levels of IgM (0.91 μ g/ml) and IgA (0.34 μ g/ml), reflecting the community immunity against Hib. Low anti-Hib level (< 0.15 μ g/ml of anti-PRP IgG) in elderly people (males aged 57–91 years and females aged 35–64 years) may indicate a need for a booster dose of Hib vaccine to elderly people in the community. The IgG prevalence over IgM, and IgM prevalence over IgA indicate the major role of IgG over IgM and IgA in keeping immunity in the track against Hib. Low level of IgM and IgA comparing to IgG may indicate the absence of Hib acute infections in the population. Show more

Keywords: Haemophilus influenzae type b (Hib), anti-polyribosyl-ribitol phosphate (PRP), conjugate vaccine, prevalence, antibodies

DOI: 10.3233/HAB-180342

Citation: Human Antibodies, vol. 26, no. 4, pp. 225-235, 2018