Surface-Independent and Oriented Immobilization of Antibody via One-Step Polydopamine/Protein G Coating: Application to Influenza Virus Immunoassay.

For the construction of high-performance biosensor, it is important to interface bioreceptors with the sensor surface densely and in the optimal orientation. Herein, a simple surface modification method that can optimally immobilize antibodies onto various kinds of surfaces is reported. For the surface modification, a mixture of polydopamine (PDA) and protein G was employed. PDA is a representative mussel-inspired polymer, and protein G is an immunoglobulin-binding protein that enables an antibody to have an optimal orientation. The surface characteristics of PDA/Protein G mixture-coated substrates are analyzed and the PDA/protein G ratio is optimized to maximize the antibody binding efficiency. Moreover, the antibody-immobilized substrates are applied to the detection of influenza viruses with the naked eye, providing a detection limit of 2.9 × 103 pfu mL-1 . Importantly, the several substrates (glass, SiO2 , Si, Al2 O3 , polyethylene terephthalate, polyethylene, polypropylene, and paper) can be modified by simple incubation with the mixture of PDA/protein G, and then the anti-influenza A H1N1 antibodies can be immobilized on the substrates successfully. Regardless of the substrate, the influenza viruses are detectable after the sandwich immunoreaction and silver enhancement procedure. It is anticipated that the developed PDA/protein G coating method will extend the range of applicable materials for biosensing.

ß-Microseminoprotein endows post coital seminal plasma with potent candidacidal activity by a calcium- and pH-dependent mechanism.

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70-75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as ß-microseminoprotein. At neutral pH, the fungicidal activity of ß-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E(71) was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of ß-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine ß-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the ß-microseminoprotein family. By electron microscopy, ß-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that ß-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify ß-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Distribution of radioiodinated estramustine binding protein antibody in mice with DU-145 prostate cancer xenograft.

BACKGROUND: Estramustine phosphate (EMP) and estramustine binding protein antibody (EMBP-AB) accumulate in the mouse prostate. The distribution of radioiodinated EMBP-AB in tumor mice was investigated to assess its therapeutic potential against prostate cancer. MATERIALS AND METHODS: Mice with DU-145 prostate cancer xenografts received 243 microCi of I-125-labeled EMBP-AB (RI-EMBP-AB). The concentration of activity in different organs was measured 4 h after the injection. RESULTS: The blood contained 0.45% of the injected dose per gram, the prostate 2.4%, the testes 0.95% and the tumor 0.65%. Radioactivity in these organs decreased more rapidly than in other organs. The doses of radiation absorbed by the prostate and the tumor, assuming a 1 mCi injected dose, were 1.81 and 0.92 cGy, respectively. CONCLUSION: Most other organs would receive relatively high doses of radioactivity, were I-125 to be used in therapeutic doses. Therefore, RI-EMBP-AB is not beneficial in radionuclide treatment as compared to possible EMP applications.

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin.

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as beta-microseminoprotein (beta-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding.

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins.

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.

Ab initio structure of human seminal plasma prostatic inhibin gives significant insight into its biological functions.

Human seminal plasma prostatic inhibin (HSPI) is a protein isolated from the human prostate gland. Despite its profound biomedical and biotechnological importance, the 3D structure of this protein of 94 amino acids remains undeciphered. The difficulties in extracting it in pure form and crystallizing it have restrained the determination of its structure experimentally. The homology-based computational methods are also not applicable, as HSPI lacks sufficient sequence homology with known structures in the protein data banks. We have predicted the structure of HSPI by a knowledge-based method using nonparametric multivariate statistical techniques. Stereochemical and other standard validation tests confirm this to be a well-refined structure. The biophysical properties exhibited by this structure are in good agreement with the NMR experimental observations. Docking and other computational studies on this structure provide significant explanation and insight into its binding activities and related biological and immunogenic functions and offer new directions for its potential applications.

Expression, specificity and immunotherapy potential of prostate-associated genes in murine cell lines.

The TRAMP-C1 (C1) and TRAMP-C2 (C2) cell lines were derived from a prostate tumor that arose in a mouse from the transgenic adenocarcinoma mouse prostate (TRAMP) model. However, their similarity to primary prostate tumors and therefore their usefulness in immunotherapy studies has not been clearly defined. We showed using RT-PCR that these cell lines exhibited a variety of prostate-specific genes expressed by human prostate tumors that may be used as tumor-associated antigens for immunotherapy. Interestingly, several of these genes are also expressed in cell lines that are not prostatic in origin. The prostate cell lines were also shown to grow in an androgen-independent manner, to be capable of expressing MHC class I and to be susceptible to specific lysis by cytotoxic T lymphocytes. Therefore, these cell lines will provide us with the ability to evaluate immune responses to and tolerance of prostate-specific protein peptides in an animal model.