Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (

Immunogencity of HSA-L7/L12 (Brucella abortus Ribosomal Protein) in an Animal Model

Background: The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. Objective: This study was aimed to evaluate the protection of recombinant Human Serum Albumin (HAS)-L7/L12 fusion protein in Balb/c mice. Methods: The amplified L7/L12 gene was cloned in pYHSA5 vector, pYHSA5-L7/L12 construct was transformed in Saccharomyces cerevisiae and the expressed protein from supernatant was purified by affinity chromatography. Balb/c mice were immunized in five groups by tHSA-L7/L12 fusion protein (group 1), Brucella abortus S19 (group 2), HSA (group 3), recombinant L7/L12 (group 4), PBS (group 5). ELISA to detect antibody production, LTT test to assess antigen specific lymphocyte response were conducted prior to virulent B. abortus strain 544 challenge two weeks after the last injection. Bacterial counts from spleens of immunized mice were done four weeks after challenge. Results: In ELISA tests, the specific antibodies exhibited a dominance of immunoglobulin IgG1 over IgG2a. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference in proliferative response of L7/L12 and tHSA-L7/L12 fusion protein (p>0.05). The L7/L12 and tHSA-L7/L12 fusion protein vaccines could also induce significant protection against challenge with the virulent strain B. abortus 544 in Balb/c mice (p <= 0.05). Conclusion: The tHSA-L7/L12 fusion protein, similar to L7/L12 has the ability to induce antigen specific lymphocyte proliferation, stimulate humoral immunity and engender protection

Protection of BALB/C mice against Brucella abortus 544 challenge by vaccination with combination of recombinant human serum albumin-l7/l12 (Brucella abortus ribosomal protein) and lipopolysaccharide

BACKGROUND: The immunogenic Brucella abortus ribosomal protein L7/L12 and Lipopolysaccharide (LPS) are promising candidate antigens for the development of subunit vaccines against brucellosis. OBJECTIVE: This study was aimed to evaluate the protection of combination of recombinant HSA-L7/L12 fusion protein with LPS in Balb/c mouse. MATERIALS AND METHODS: The recombinant HSA-L7/L12 fusion protein in Saccharomyces cerevisiae was expressed and purified by affinity chromatography column. LPS was extracted by n-butanol, purified by ultracentrifugation. BALB/c mouses were immunized in 9 groups with PBS, HSA, tHSA-L7/L12, L7/L12, LPS, LPS+ HSA, LPS+ tHSA-L7/L12, LPS+ L7/L12, B. abortus S19. ELISA, LTT tests and challenging two weeks after last injection were carried out. Bacterial count of spleen of immunized BALB/c mouse was done four weeks after challenging with virulent strain B. abortus 544. RESULTS: In ELISA test the specific antibodies of tHSA-L7/L12 exhibited a dominance of immunoglobulin IgG1 over IgG2a. LPS-HSA and tHSA-L7/L12 + LPS produced a significantly higher antibody titer than LPS alone and L7/L12+LPS (P 0.05). The combination of tHSA-L7/L12 fusion protein with LPS and B. abortus S19 induce higher level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice than other groups (P = 0.005). CONCLUSIONS: The combination of tHSA-L7/L12 fusion protein with LPS had higher protective ability than LPS and fusion protein distinctly

Radioimmunoscintigraphy of Breast Tumor Xenografts in Mouse Model by 99mTc Direct Radiolabeling of a Monoclonal Antibody PR81

Introduction: The radioimmunoscintigraphy (RIS) has found widespread clinical applications in  tumor  diagnosis.  Human  epithelial  mucin,  MUC1,  is  commonly  over  expressed  in  adenocarcinoma including 80% of breast cancers and represents a useful target for RIS. The PR81  is  a  new  murine  anti-MUC1  monoclonal  antibody  that  was  found  to  react  with  the  membrane  extracts of several human breast cancerous tissues and the cell surface of some MUC1 positive  cell lines. In this study, a direct method which is very simple, rapid and efficient for the labeling  of this MAb with  99m Tc, particularly suitable for the development of a ‘kit’, was developed. The  quality  control  of  new  radiopharmaceutical  and  immunoscintigraphy  studies  in  BALB/c  mice  bearing breast tumor xenografts were also performed.  Materials and Methods: The Ab reduction was performed with 2-mercaptoethanol (2-ME) at a  molar  ratio  of  2000:1  (2-ME:MAb)  and  reduced  Ab  was  labeled  with  99m Tc  via  methylene  diphosphonate (MDP) as a transchelator. The labeling efficiency was determined by ITLC. The  amount  of  radiocolloids  was  measured  by  cellulose  nitrate  electrophoresis.  The  stability  of  the  labeled product was checked in fresh human serum by gel filtration chromatography (FPLC) over  24 hrs. The integrity of the labeled MAb was checked by the means of SDS-PAGE. Cell-binding  assay  was  used  to  test  the  binding  ability  of  99m Tc-PR81  to  MCF7  cells.  Biodistribution  was  studied in normal BALB/c mice at 4 and 24 hrs post-injection. The tumor imaging was performed  in female BALB/c mice with breast tumor xenografts 24 hrs after the new complex injection.  Results:  The  labeling  efficiency  was  94.2%±2.3  and  radiocolloids  were  2.5%±1.7.  In  vitro  stability  was  70%±5.7  in  fresh  human  serum  over  24  hrs.  There  was  no  significant  Ab  fragmentation due to the labeling procedure. Both the labeled and unlabeled PR81 were able to  compete for binding to MCF7 cells. The biodistribution studies in normal BALB/c mice showed  that  there  was  no  important  accumulation  in  any  organ.  The  immunoscintigraphy  studies  demonstrated definite localization of the preparation at the site of tumors with high sensitivity.  Discussion and Conclusion: The results show that by using the Schwarz method of radiolabeling  MAb PR81, a labeling yield higher than 90% with high stability of the complex in human serum  can be obtained. These findings demonstrated that the new radiopharmaceutical can be considered  as a promising candidate for imaging of human breast cancer