Hugh J. Callahan

[2] Proteins and polypeptides as antigens

Publisher Summary This chapter discusses the role of proteins and polypeptides as antigens. Antibodies against enzymes can be used to detect and assay quantitatively the concentration of enzymes; to concentrate and purify the enzymes from dilute solutions and mixtures; to study the active catalytic sites, multimolecular forms, and conformational structures of the enzymes; to localize the enzymes in sectioned cells; and to study the appearance and modification of the enzymes in the course of embryonic and phylogenetic development. Concomitantly, in immunology, there has been increasing knowledge concerning many factors that can influence the multifaceted and complex sequence of events of the immune response, beginning with the introduction of an antigen into a host (immunogen) to the formation of humoral antibody. This chapter discusses the information about techniques that have become available for producing antibody. It also attempts to make an investigator aware not only of factors that might enhance antibody formation, but also of factors that might be operative in the suppression of the immune response.

Antisperm antibodies in seminal plasma of spinal cord-injured men.

Central to the problem of reproductive rehabilitation of spinal cord-injured men treated by assisted ejaculatory techniques is the consistent observation of deficient semen quality. Most studies have reported asthenospermia despite the presence of normal sperm concentration in most men undergoing these procedures. To date little attention has been given to the incidence and relevance of sperm autoimmunity in this group. In 9 anejaculatory spinal cord-injured men, electroejaculation was performed. Antegrade ejaculates were obtained in 7 men and analyzed. Mean sperm antegrade concentration was 74.4 +/- 113 x 10(6)/mL with a mean motile sperm concentration of 28.6 +/- 54.0 x 10(6)/mL. Enzyme-linked immunosorbent assay (ELISA)-determined antisperm antibody response was positive in the seminal plasma of 5 of 7 patients. Because of the disproportionately high incidence of an immunologic factor in men with neurogenic infertility, sperm autoimmunity should be considered among the important causes underlying their seminal dysfunction.

Urinary Bladder Glycoproteins of the Rabbit: Extraction, Biochemical and Immunological Studies

The mucin layer of the bladder covering the transitional epithelium is thought to be an anti-adherence substance for bacteria. We have previously demonstrated that removal of this layer results in increased bacterial colonization. In this present communication we report our attempts to isolate and characterize the components of the mucin layer. Bladders were removed from female NZW rabbits and the mucosal layer was extracted with 0.1 M NaCl. Fractional centrifugation and column chromatography on AcA 34 and AcA 22 resulted in the separation of a partially purified glycoprotein, containing 30% carbohydrate, migrating as a single peak (4.5S) in the analytical ultracentrifuge. It was immunogenic in mice and the antisera were used to develop both a radioimmunoassay and an enzyme linked immunosorbent assay (ELISA). The antigenic activity of the glycoprotein was destroyed by protease or extremes of pH, but not by several glycosidases. In addition the murine antisera could not be inhibited by a panel of naturally occurring glycoproteins or glycosaminoglycans.

Systemic sperm autoimmunity in spinal-cord injured men

Historically, spinal-cord injured men have been considered virtually sterile because of ejaculatory dysfunction commonly resulting from their injury. Assisted ejaculatory techniques, however, have overcome the problem of sperm transport and have allowed both the establishment of pregnancy through artificial insemination and the assessment of their semen quality. Most studies have noted the presence of asthenozoospermia in the setting of normal sperm concentration following electroejaculation or vibratory stimulated ejaculation. Thus far, little attention has been given to the basis for the frequent finding of asthenozoospermia, and the possibility of sperm autoimmunity in this group has not been adequately studied. In nine spinal-cord injured men, reproductive evaluation was performed consisting of hormonal measurements, testicular biopsy, and indirect immunobead tests for sperm autoimmunity. A mean sperm concentration was 144 +/- 185 x 10(6)/ml. However, the mean motile concentration was 33 +/- 62 x 10(6)/ml. Indirect serum immunobead showed positive IgG or IgA titers in 3 of 8 patients. Because of the disproportionately high incidence of an immunologic factor in spinal-cord injured men compared to able-bodied infertile men, sperm autoimmunity should be considered among the important causes underlying seminal dysfunction following spinal cord injury.

Preparation of an antigen-binding fragment from murine T lymphocyte membranes

Abstract BALB/c mice were immunized with the random sequence polypeptide [Glu 60 Ala 40 ] n (GA). ∗ Spleen and lymph nodes were used to prepare T lymphocyte suspensions via nylon-wool columns. These cells were radiolabelcd with 125 I by the lactoperoxidase procedure and then ruptured by nitrogen cavitation. Membrane fragments were isolated by sucrose density centrifugation and subsequently solubilized in Triton X-100. Nonspecific material was removed by passing the solubilized membranes through an immunoadsorbent composed of d -GA (i.e. [ d -Glu 60 - d -Ala 40 ] n ) covalently bound to Sepharose 4B. The nonadherent fraction was subsequently passed through l -GA Sepharose and the adherent material eluted with 3 M NaCNS. After removal of salt and relabeling, the eluate was readsorbed onto and eluted from a fresh l -GA column. This material bound almost completely to l -GA immunoadsorbents (80–85%) but at control levels (8–10%) to immunoadsorbents composed of d -GA, d -GAT (i.e. [ d -Glu 60 - d -Ala 30 - d -Tyr 10 ] n ) or l -GAT. Polyacrylamide gel electrophoresis revealed only one component with a molecular weight of 60,000–70,000. Gel filtration chromatography in the presence of Triton X-100 indicated a higher molecular weight, probably due to aggregation and/or detergent binding. Membranes were also prepared from normal, non-immune BALB/c mice and fractionated over immunoadsorbent columns in the manner described. On the basis of incorporated radiolabel, the total amount of antigen-binding membrane recovered was 15% of that obtained in immune animals. These membranes demonstrated no specificity in binding to either l -GA or d -GA immunoadsorbents.

Antisera to a rabbit urinary tract antigen also react with human bladder and kidney tissue.

The mucin layer covering the bladder transitional cell mucosa appears to function as a primary defense mechanism against bacterial infection. We have previously prepared a glycoprotein fraction (GP1) from the urinary bladder mucosa of NZW rabbits and raised murine antisera against it. These antisera react with bladder, ureter and kidney tissue from rabbits, rats, guinea pigs, and hamsters. We now show that a similar substance occurs in human kidneys and bladder. In order to remove antibodies reactive with the Tamm-Horsfall protein (THP), the antisera were initially absorbed with an immunoadsorbent composed of purified human THP covalently bound to Sepharose CL-4B gel. Using an enzyme linked immunosorbent assay (ELISA) it could be shown that the absorbed antisera did not react with THP but retained a high titer in binding to GP1. Immunohistochemical procedures involving avidin-biotin-immunoperoxidase staining demonstrated that the absorbed anti-GP1 reacted well with six human urinary bladder biopsy specimens and two kidney autopsy specimens while normal murine sera showed little or no binding. Although this reactivity was not as strong as that found with homologous tissue (rabbit) these studies suggest that GP1, an antigen common to several animal species, is also related to a human urinary tract component.

Urinary tract glycoprotein: distribution and antigenic specificity

SummaryIt has previously been demonstrated that the urinary tract contains a unique glycoprotein (GP1) that appears to serve a function in the clearance of pathogenic bacteria. We prepared a panel of monoclonal antibodies (mAbs) to human GP1 and examined its antigenic specificity and distribution in the human urinary tract. This was also observed in relation to another glycoprotein associated with infection, Tamm-Horsfall protein (THP), as well as to URO-5, a protein used in identifying the lower urinary tract. In enzyme-linked immunoadsorbent assays (ELISA), all of the GP1 mAbs reacted with GP1 prepared from both human urine and rabbit bladder mucosa. No immunoreactivity was observed with either THP mAbs or URO-5 mAbs. Western-blot analyses using GP1 mAbs with human urine GP1 preparations detected a single band of approximately 50–52 kDa. Human urinary tract tissues were also examined by immunohistochemical techniques using the GP1 mAbs, commercial THP, and URO-5 mAbs. In paraffin-embedded bladder sections, only GP1 mAbs reacted with the urothelium. Selective staining of distal collecting tubules, renal pelvis, and ureters was demonstrated for GP1 mAbs. Proximal convoluted tubules, loops of Henle, and Bowmans capsule failed to stain for GP1. In the same tissues, THP mAbs reacted with the thick and thin segments of medullary loops of nephrons (Henles loops). URO-5 mAbs stained fresh frozen sections of bladder urothelium, distal collecting tubules, and portions of Henles loops; however, they failed to stain paraffin-embedded tissue sections. These GP1 mAbs provide a new tool for biochemical and histochemical investigations of the mucin layer in both healthy and diseased urinary tract tissue.

Distribution of rabbit mucosal glycoprotein throughout urinary tract

The mucin layer covering the transitional epithelium of the bladder is thought to be an anti-adherence substance for bacteria. We describe the use of an immunoperoxidase staining technique to demonstrate the presence of glycoprotein lining the urothelium of both the upper and lower urinary tracts of the rabbit. Antisera against this glycoprotein (GP1) were raised in Swiss-Webster mice. The genitourinary tracts of male and female NZW rabbits were removed and sequentially treated with mouse anti-GP1 sera, biotin-labelled anti-mouse IgG, and an avidin-biotinylated horseradish peroxidase complex. The results demonstrated that an antigenically similar (or identical) glycoprotein covers the distal renal tubules and urothelium of the renal pelvis, ureters, bladder, and urethra, suggesting that it may function as an antibacterial defense mechanism throughout the urinary tract.

Antigenic Similarities among Rodent Urinary Tract Glycoproteins

Urinary glycoconjugates (glycosaminoglycans, glycoproteins, mucopolysaccharides) have been postulated as the natural defense mechanisms which prevent urinary tract infections. As a direct approach to establish the validity of this hypothesis, we have prepared a glycoprotein fraction (GP1) from rabbit bladder mucosal tissue and shown that it may be involved in the prevention of bacterial adherence. Immunohistochemical studies using fluorescence have demonstrated that murine antibodies raised to rabbit GP1 can be used as a semiquantitative index of glycoprotein production. The present investigation addresses the question of the species restriction of the glycoprotein--whether it is confined to the rabbit or whether a similar or identical substance occurs in other species. Using an immunoperoxidase staining technique, we have examined the genitourinary tracts of Sprague Dawley rats, Golden hamsters, and Hartley guinea pigs. Mouse anti-rabbit GP1 was used as the primary antibody with an avidin-biotin complexing system. All three species reacted well with the murine antiserum but were negative with normal sera. Semiquantitative estimates of the relative amounts of this material in different areas of the genitourinary tract showed that the distal renal tubules, renal pelvic mucosa, ureters and bladders were rich in this glycoprotein while urethra and vagina were not.

Physicochemical Studies of Ca ++ Controlled Antigen Antibody Systems

In this paper we will briefly discuss some basic aspects of immunoglobulin structure and function. We will then present in some detail our studies on the divalent cation controlled antigenantibody reactions which are germane to this Symposium. And finally, we will describe some of the fundamental unresolved problems concerning the antibody molecule and show how we have used these reactions to investigate some of these problems.