Bonnie S. Dunbar

[51] Protein blotting and immunodetection

Publisher Summary This chapter discusses the procedure for protein blotting and immunodetection. The choice of buffer composition depends on the types of gel and membrane selected. The procedure of Towbin as modified by Anderson specifies a Tris-glycine pH 8.3 buffer containing sodium dodecyl sulfate (SDS). The recirculating, ice-cooled, high ionic strength buffer used helps prevent the gel from swelling in the absence of methanol during transfer, which can cause poor resolution of the proteins on the membrane. Transfer membrane, four sheets of filter paper, and two foam pads are cut to the same size as the gel and soaked in electrode buffer. If the buffer warms during the procedure, a recirculating cooling bath may be needed. After transfer is complete, place the membrane into a tray which is slightly larger than the sheet itself to ensure efficient mixing of solutions over the paper. The side of the paper that is next to the gel must be placed facing up. The use of prestained markers help to determine the side on which the proteins are immobilized.

Preparation of polyclonal antibodies.

Publisher Summary The term “polyclonal antibody” is defined as the total population of antibodies present in animal serum. This complex population contains different antibody subclasses including IgG, IgM, IgE, IgA, and IgD. The term “immunogen” refers to any molecule that is capable of eliciting an immune response. Thus, immunogenicity refers to the level of immune response elicited. It is necessary to obtain sufficient quantities of the highly purified immunogen that will be used to immunize the animal. In a protein preparation that contains as little as 1% contamination, the majority of antibodies may recognize that contaminant if it is highly immunogenic. The use of two-dimensional gel electrophoresis has revolutionized the ability to easily isolate denatured proteins or peptides, which are sufficiently purified to generate specific polyclonal antibodies. In addition, new products have been designed to isolate IgG by affinity chromatography using protein A attached to cellulose disks. These may prove to be useful for some applications.

Formation of the rabbit zona pellucida and its relationship to ovarian follicular development

The zona pellucida is the unique extracellular glycoprotein matrix which is assembled during growth of the mammalian oocyte. The present studies were carried out to examine the formation of this structure in relation to the differentiation of ovarian cell types during follicular development. Specific antibodies were developed against total rabbit ZP proteins as well as against ZP proteins electrophoretically purified by high-resolution two-dimensional polyacrylamide electrophoresis gels (2D-PAGE). Antibodies were characterized by (a) immunoelectrophoresis, (b) a Staphylococcus aureus protein A binding assay, and (c) immunoblotting following 2D-PAGE separation of ZP proteins. Immunoperoxidase localization with these antibodies was used to determine the stage of ovarian follicular development at which ZP antigens first appear as well as to evaluate the cellular and extracellular distribution of these proteins throughout folliculogenesis. The ZP proteins were first observed in the cytoplasm and at the periphery of the oocytes surrounded by a thin squamous follicular cell layer. No staining was observed in the cytoplasm of follicle cells during early folliculogenesis. As the ZP matrix was assembled extracellularly, the intensity of staining of the outer and inner regions could be distinguished. This differentiation of the matrix coincided with the differentiation of the follicular cells into a multilayer cell complex. At this stage, specific ZP proteins are localized within the cytoplasm of the inner layers of these follicular cells. The staining is then diminished in cells of preantral follicles. These studies demonstrate that the formation of the ZP is an excellent model system to study the early stages of follicular development and cell differentiation.

Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system.

A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a baculovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm‐ZP interaction. The expressed glycosylated protein, BV55, was purified by wheat germ agglutinin lectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP‐α‐BV55) is specific for the 55 kDa rabbit ZP protein. Indirect immunofluorescence studies indicate that GP‐α‐BV55 localizes to a filamentous meshwork on the surface of the ZP of isolated rabbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrated that this antigen is localized within the ZP of primary and more advanced stage ovarian follicles but is not detected in primordial follicles. In addition, the 55 kDa antigen was detected in the granulosa cells of secondary stage follicles but not in the oocyte. GP‐α‐BV55 effectively blocked the binding of rabbit sperm to rabbit eggs in vitro. However, Fab fragments generated from GP‐α‐BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP‐α‐BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding, additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with ligand activity in the sperm‐ZP interaction, and that BV55 bound to rabbit sperm in a dose‐dependent manner. These studies therefore demonstrate that antibodies against recombinant ZP proteins recognize the native intact ZP and inhibit sperm‐ZP interaction. They also provide evidence that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3α sperm receptor protein, has sperm receptor activity.

Protein analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis

Publisher Summary This chapter describes the use of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein analysis. The use of 2D-PAGE has become increasingly popular during the past decade. 2D-PAGE allows the resolution of a complex protein mixture into more discrete components than 1D-PAGE because it separates on the basis of protein charge in addition to molecular weight. The most common uses of 2D-PAGE are the analysis of complex mixtures of proteins and the analysis of the posttranslational modification of proteins. 2D-PAGE can also provide valuable information about the molecular properties of proteins, including an estimate of the relative isoelectric points (p I ) and molecular weights of proteins. The preparation of samples for 2D-PAGE analysis is the most critical step in guaranteeing excellent reproducible results. If standard electrophoresis chambers are used, electrophoresis is carried out by placing the electrode buffer in the upper and lower chambers. The slab gels are then placed into these chambers, taking care to avoid air bubbles being trapped at the bottom of the slab acrylamide gel.

Comparative Structure and Function of Mammalian Zonae Pellucidae

The process of fertilization, like other aspects of mammalian reproduction, varies markedly among different species. Of the many complex events that accompany this process, the interaction between the sperm and the zona pellucida (ZP) is one of the most significant. The mammalian ZP is an extracellular glycoprotein structure that is formed around the oocyte during the early stages of ovarian follicular development. This matrix is important in the initial stages of fertilization because the sperm must bind to and penetrate it before fusing with the oocyte plasma membrane (Austin and Braden, 1956; Austin, 1975). The ZP is also involved in the block to polyspermy in many species and further serves to protect the embryo after fertilization until implantation in the uterine wall. Species differences have been reported for several steps in the fertilization process, including sperm capacitation (the changes spermatozoa undergo in the female reproductive tract in order to develop the capacity to fertilize the egg) and sperm binding to and penetration of the ZP. Many of these differences can be attributed to morphological and biochemical variations of the gametes themselves (Yanagimachi, 1977; Bedford, 1974; other chapters in this text).

Zona pellucida antigens: targets for contraceptive vaccines.

The mammalian ovary is unique among secretory organs in its ability both to produce the female gamete and to provide the necessary endocrine support for its orderly development. This set of functions utilizes complex feedback mechanisms involving the hypothalamus and the anterior pituitary over the reproductive life of the individual. The late stages of follicular development just prior to ovulation have been the focus of most ovarian studies. However, it is necessary to investigate the earliest stages of follicular development in order to begin to understand the foundations of immunologically based interference with ovarian function.

Molecular Analysis of a Carbohydrate Antigen Involved in the Structure and Function of Zona Pellucida Glycoproteins

Abstract A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-β-glycosidase, endoglycosidase F, or combinations of neuraminidase plus β-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.

Use of immunoaffinity purified antibodies to zona pellucida to compare alloimmunization of male and female rabbits

In order to study the immunogenicity as well as tissue specificity of zona pellucida (ZP) antigens, the present studies have been designed to examine the effects of alloimmunization of male and female rabbits with rabbit zonae pellucidae. These studies are the first to demonstrate that high titers of antibodies to homologous ZP antigens are developed in male rabbits while no detectable antibodies are developed in females. As demonstrated using the ELISA assay, the antibodies from these males immunized with rabbit ZP, have a greater reactivity against rabbit ZP antigens than do antibodies from female rabbits heteroimmunized with porcine ZP. The antibodies from the male rabbits immunized with rabbit ZP also recognize antigenic determinants of porcine ZP. Methods for the immunoaffinity purification of antibodies from serum were developed to determine whether low levels of antibodies against ZP are present in sera of alloimmunized female rabbits. They also allow more detailed analysis of antibodies used to detect antigenic determinants which are cross-reactive between different mammalian species. Although this method was effective in isolating low levels of antibodies from male alloimmunized rabbits or from female rabbits heteroimmunized with porcine ZP proteins, no specific antibodies could be isolated from the serum of females alloimmunized with rabbit ZP. These studies more clearly demonstrate that zona pellucida antigens are specific to the ovary in that female rabbits do not develop significant antibody levels against rabbit ZP antigens, even following active immunization with adjuvant, while male rabbits develop high titers of antibodies.