Production and characterization of monoclonal antibodies against human prostate specific antigen

Prostate Specific Antigen [PSA] is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies [mAbs] against PSA have been presented. Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer [PC alpha], Benign Prostatic Hyperplasia [BPH] and brain cancer tissues by Immunohistochemistry [IHC]. Five anti-PSA mAbs [clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/K] and clones [2C8-E9, 2G3-E2, IgG2 alpha/K] were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kD alpha in human seminal plasma in western blot. These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids

Isolation and partial characterization of human amniotic epithelial cells: the effect of trypsin

Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells [hAECs] remains controversial. The aim of the present study was to characterize immunophenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor [EGF]. To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC

Production and characterization of a marine monoclonal antibody against human ferritin

Ferritin is an iron storage protein, which plays a hey role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody [mAb] against human ferritin was reported. Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma [clone: 2F9-C9] was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity [2.34 10[9] A/[1]] and the isotype was determined to be lgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic bit if other requirements of the hit are met

Comparison of the photobleaching and photostability traits of alexa fluor 568- and fluorescein isothiocyanate- conjugated antibody

Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate [FITC] and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody [ANM] to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry [ICC]. Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC

Conjucation of R-Phycoerythrin to a polyclonal antibody and F [ab'] 2 fragment of a polyclonal antibody by two different methods

R-phycoerythrin [R-PE], a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F[ab']2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry [ICC] and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis [SDS-PAGE]. In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity

Conjugation of monoclonal antibodies to super paramagnetic iron oxide nanoparticles for detection of her2/neu antigen on breast cancer cell lines

Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide [SPIO] nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20microg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques

Assessment of thyroglobulin expression in reproductive organs at different stages of mouse estrous cycle

Prevalence of abortion is higher in women with autoimmune thyroid disease. In the majority of cases, however, no abnormality of thyroid function is detected despite the high levels of antithyroid antibodies. The direct influence of such harmful autoantibodies in female reproductive organs may serve a role in pregnancy loss. In this study, expression of thyroglobulin in the reproductive tissues of cycling mice has been evaluated. Stages of estrous cycle were determined by cellular morphology and ratio of epithelial cells to leukocytes in vaginal smear of Balb/C mice. At each phase, the mice were sacrificed and their uterus, ovary and fallopian tubes were removed. Expression of thyroglobulin-specific transcript in endometrium was investigated by two sets of primers using reverse transcriptase-polymerase chain reaction [RT-PCR]. In addition, expression of thyroglobulin in reproductive tissues was assessed by immunohistochemistry and dot blot analysis. The results showed that thyroglobulin mRNA is not expressed in endometrial tissue of Balb/C mice at any stage of estrous cycle. Immunohistochemical analysis also confirmed that thyroglobulin or its cross reactive-antigens are not expressed at the protein level in the female reproductive organs. The results showed that thyroglobulin was not expressed in the reproductive organs of female mice. It is plausible that antithyroglobulin antibodies could interact with newly-generated antigens during placentation and pregnancy

Production and characterization of murine monoclonal antibodies recognizing conformational and linear epitopes localized on human IgA2 molecules

There are two subclasses of human IgA [IgA1 and IgA2] that differ in antigenic properties and in chemical composition. The constant domains of alpha1 and alpha2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies [MAbs] specific for conserved conformational or linear epitopes restricted to each subclass. To produce, select and characterize monoclonal antibodies specific for human IgA2. Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine [HAT] selective medium and cloned by limiting dilution assay. Antibody [Ab] secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant [K[aff]] was also determined by ELISA. Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope[s] while 2F20B5 and 3F20E3 react with conformational epitope[s] located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested. These MAbs, especially 6F20H11 with high affinity constant [6.03 x10[9] M[-1]] are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animal sera suggests phylogenic conservation of the epitope recognized by this MAb