Dual specificity antibodies using a double-stranded oligonucleotide bridge

AbstractThe covalent conjugation of oligonucleotides to antibody Fab’ fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics

Investigation into the characteristics and properties of prostasomes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Seminal hemostatic factors: then and now

Human semen spontaneously coagulates and subsequently liquefies following ejaculation. The exact reasons for this phenomenon are not entirely understood. Research has resulted in the discovery of several seminal proteins that are already known from another major domain of human physiology (i.e., blood coagulation and fibrinolysis). Some believe that regulation of seminal coagulation and fibrinolytic liquefaction may bear analogies to the well-described pathways operational in blood. We here attempt to summarize the subject, revisiting earlier findings and covering the more recent and extensive data on hemostatically functional seminal proteins, including the findings of our own group. Evidence for the existence of each key hemostatic factor in semen is presented along with any correlation with indices of male fertility. Several probably possess alternative functions including anti-inflammatory action, aiding sperm capacitation and classic hemostasis following intercourse-induced bleeding. Established mechanisms such as the high molecular weight seminal vesicle system are also considered as potentially interacting with conventional hemostatic pathways to regulate seminal coagulation and liquefaction. Although our grasp of this subject continues to evolve, there still remain many unanswered questions. A more complete understanding may one day prove useful in designing technologies for improved diagnostics in male infertility and potentially aiding assisted human reproductive therapie

Prostasomes, angiogenesis, and tissue factor

Prostasomes are membrane-bound secretory vesicles produced by prostatic epithelial cells. They are known to carry many proteins, including tissue factor, and have membranes unusually rich in cholesterol and sphingomyelin. Prostasomes have well-documented effects on fertility, promoting sperm motility, stabilizing the acrosome reaction, and facilitating immunosuppression. This article reviews the evidence of the effects of prostasomes on in vitro angiogenesis assays, and the mechanism by which these effects occur. Seminal prostasomes seem to inhibit angiogenesis, whereas the equivalent particles released by malignant prostate cells promote angiogenesis. In both cases, the effects seem preserved after heat treatment to denature the protein content, suggesting an important role for lipid transfer, in particular, transfer of sphingomyeli

Anti-prostasome antibodies are not an appropriate prognostic marker for prostate cancer

Objective Antiprostasome antibodies (APAs) have been identified in serum of patients with prostate cancer and have been proposed as a new marker for metastatic disease. This study reassesses the role of APAs as a prognostic indicator for prostate cancer. Material and methods Serum samples from healthy controls (n7) and patients with prostate cancer (n22) were assayed for APAs using an enzyme-linked immunosorbent assay. Results APAs in varying amounts were present in healthy individuals as well as in men with prostate cancer. Higher levels were inversely and significantly associated with prostatespecific antigen (PSA). No significant relationships were noted between APA levels and other parameters such as age, time since diagnosis, metastatic status, Gleason histological score and hormonal treatment. Conclusions The presence of serum APA is unlikely to be a strong prognostic indictor for prostate cancer on an individual basis as false positives will occur. However, such immune reactions which may be associated with PSA in cancer patients are in any case of interest in both the biology of prostate cancer and male fertility. The source of prostasomal antigen may be of critical importance to the outcome of the assay. However, immune reactions to prostasomes may be of considerable interest and warrant continued investigation

Seminal factor VII and factor VIIa: Supporting evidence for the presence of an active tissue factor dependent coagulation pathway in human

Human semen spontaneously coagulates into a semisolid mass and then wholly liquefies in a process that may have some similarity to that of normal blood. This well described phenomenon is referred to as coagulation and liquefaction of semen. Besides other active components of the haemostatic system, semen contains a significant amount of functional tissue factor (TF). However, TF needs factor (F)VII in order to exert it actions. In this study, we assessed human semen for the presence of FVII and FVIIa, and related their levels to conventional fertility parameters. Using a functional, one stage, clotting assay based upon the prolongation of the prothrombin clotting time, using the ACL 300R analyser and an Imubind® FVIIa ELISA assay, FVII and FVIIa levels were measured in 97 semen specimens obtained from sub-fertile (sperm counts <20 × 106/mL), normally fertile (sperm counts ?20 × 106 but <60 × 106/mL), fertile sperm donors (sperm counts ?60 × 106/mL), vasectomized subjects and in a pooled normal semen parameters group (categorization into groups was based on the World Health Organization guidelines on fertility criteria). In addition, conventional semen parameters were analysed on all semen samples. Both FVII and FVIIa were quantifiable in human semen. The mean levels of FVII and FVIIa were 4.4 IU/dL and 12 ng/mL respectively. Despite the observed variations of FVIIa levels in the studied groups they did not meet statistical significance when the groups were tested against each other. However, seminal FVIIa levels showed a significant positive association with semen liquefaction time, sperm motility and semen volume. The anti-sperm antibodies and sperm-agglutination groups were also associated with raised seminal FVIIa levels. We observed no significant relationship between FVIIa levels and total sperm concentration, sperm count per mL (sperm density), sperm progression and days of sexual abstinence. This study demonstrates that human semen contains appreciable amounts of FVII and FVIIa. It is possible to quantify these using commercially available assays. There also appears to be a direct correlation between the levels of these factors and certain seminal parameters. This finding reinforces the concept of an active clotting system in human semen, by establishing the missing link in the activation of a TF-dependent pathway

Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model

We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule