Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment) and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims

Purification And Characterization Of Cell Wall Mannoproteins Of Candidaalbicans Using Intact Cell Method

Virulence of the opportunistic yeast, Candida albicans , involves the interplay of many complex changes including the yeast-hyphae transition, which mainly involves protein changes. Cell wall mannoproteins are found to be the main cause of adherence of C. albicans to epithelial cells in the first step of an infection process. In the present study, cell wall mannoproteins of intact yeast were purified using a simple treatment of yeast with mercaptoethanol and sodium dodecyl sulfate followed by Concanavalin A chromatography. Both electrophoretic analysis of the column effluent and Western blot analysis using polyclonal and monoclonal antibodies showed the presence of mannoproteins with molecular weight in the range of 30-50 kDa. Dot blot analysis of the purified antigen with the polyclonal and monoclonal antibodies prepared in this study showed that outer membrane mannoprotein antigens were obtained successfully following the above simple purification strategy

The reactivity of human serum natural autoantibodies with certain autoantigens increases along with aging

Introduction Natural autoantibodies (NAAs) that recognize autoantigens are mainly generated in the absence of any apparent immunization with foreign antigens (Ags). These antibodies maintain the body homeostasis through functions, such as inhibition of tumor angiogenesis, detection of structural changes in autoantigens, and exertion of anti-inflammatory impacts. The human body is shown to go through immunological and physiological changes during aging. However, data regarding the potential variations in the binding activity of NAAs is still scarce. Therefore, in this study, we were about to explore the trend through which the reactivity of serum NAAs with several autoantigens varies with advancing age. Materials and methods Serum samples were prepared from healthy individuals of seven age intervals: (0) cord blood, (1) infancy, (2) childhood, (3) adolescence, (4) early adulthood, (5) middle adulthood, and (6) late adulthood (the elderly). The mean immune reactivity (MIR) of the sera with 24 human autoantigens that were obtained from Immunculus research center (Russia) was determined using ELISA and inter-group comparisons were also performed. Results In general, the MIR of serum natural antibodies with the autoantigens was shown to follow an upward trend with advancing age so that the lowest and highest MIRs were detected in the cord blood and late adulthood samples, respectively. Moreover, the results of the inter-group comparisons indicated that the MIRs of the first, second, fourth, and sixth groups were significantly higher than those of their previous groups, i.e. zero, first, third, and fifth groups, respectively. Conclusion This study showed that the reactivity of human tissue-specific and non-specific NAAs with autoantigens varied along with aging. Regarding the crucial roles NAAs play in maintaining the body homeostasis, the variation in their concentration at different age intervals might account for the immunological and pathological changes that occur in the elderly. © 2013 Published by Elsevier Masson SAS

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