Z. Suzanne Zam

Anti-rhodopsin monoclonal antibodies of defined specificity: Characterization and application

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsins cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsins amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsins carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

The Pathogenesis of Contact Lens-associated Pseudomonas aeruginosa Corneal Ulceration I. The Effect of Contact Lens Coatings on Adherence of Pseudomonas aeruginosa to Soft Contact Lenses

The adherence of a strain of Pseudomonas aeruginosa to hydrophilic contact lenses was quantitatively determined using scanning electron microscopy. Soft contact lenses were incubated for 72 h in either phosphate-buffered saline (PBS), a 500-μg/ml solution of human serum albumin (HSA), a 500-μg/ml solution of bovine submaxillary gland mucin, or a solution containing 500 μg/ml of both HSA and mucin. Lenses were then incubated for one hour in a 107-ml suspension of P. aeruginosa. The number of adherent bacteria per field at × 8,000 magnification was determined. Only 1.96 ± 2.52 bacteria/field were found on lenses incubated in saline, whereas 5.23 ± 5.30 bacteria/ field were adherent to lenses incubated in HSA, 23.42 ± 31.01 to lenses incubated in mucin, and 26.76 ± 35.12 to lenses incubated in a solution containing both HSA and mucin. Contact lens coatings, especially mucin, facilitate the adherence off. aeruginosa to soft contact lenses and may therefore play a role in the pathogenesis of contact lens-associated pseudomonal keratitis.

Adherence properties of Pseudomonas pili to epithelial cells of the human cornea.

In vitro adherence of Pseudomonas fluorescens organisms to the human cornea is correlated with bacterial pili. The role of pili in the attachment to human corneal epithelial cells was studied in an in vitro adherence assay system. A homogeneous, purified pilin preparation showed a molecular weight of 16,500 on SDS-polyacrylamide gels. Within 5 minutes incubation, 5.5 pg of pilin adhere to 15,000 epithelial cells. When epithelial cells were preincubated with purified pilin, a subsequent decrease in adherence of labeled pilin was noted. It appears that pili mediate adherence of Pseudomonas organism to human cornea.

The Effect of Enzymatic Contact Lens Cleaning on Adherence of Pseudomonas aeruginosa to Soft Contact Lenses

Previous studies have demonstrated that Pseudomonas aeruginosa adheres more readily to soft contact lenses with a mucin coating than to unworn contact lenses. The mucin coatings that develop on soft contact lenses may, therefore, play a significant role in the pathogenesis of contact lens-associated Pseudomonas corneal ulceration. We tested the ability of a variety of enzymatic contact lens cleaners and other enzyme solutions to decrease the adherence of Pseudomonas to mucin-coated soft contact lenses. Of the commercially available solutions that were tested, cleaning with Optizyme and Extenzyme significantly reduced the adherence of Pseudomonas to the lenses, whereas cleaning with the Softmate Weekly Cleaning System had no effect. Optizyme and Extenzyme were as effective as a 10% solution of acetylcysteine and more effective than a 0.25% trypsin solution. Neuraminidase at pH 5 was the most effective solution at reducing the adherence of Pseudomonas to the lenses, supporting the finding that sialic acid is a specific receptor for Pseudomonas aeruginosa. Soft contact lenses should be cleaned frequently with an effective enzymatic cleaner to reduce the likelihood of Pseudomonas adhering to the lens and thereby reduce the incidence of Pseudomonas corneal ulceration in soft contact lens wearers.

Failure of systemically administered adenine arabinoside to affect humoral and cell-mediated immunity.

The effect of a high dosage (250 mg/kg of body weight) of adenine arabinoside or ara-A (9-beta-D-arabinofuranosyladenine) on humoral immunity was studied in New Zealand white rabbits infected with the McKrae strain of herpes simplex virus. The rabbits were treated daily for 14 days with subcutaneous injections of ara-A. The primary and secondary humoral responses, as measured by neutralizing antibody titers, developed similarly in control and treated groups. Similar drug treatment was used on guinea pigs before or after sensitization with BCG vaccine. Subsequent challenge of the sensitized animals with Old tuberculin solution indicated that ara-A treatment had no effect on the induction or previously established cell-mediated immunity. The lack of immunosuppressive activity of ara-A at dosage levels higher than those used in primates makes this drug a potentially effective agent in the systemic treatment of herpetic infections.

A practical method for rescuing desired hybridomas during monoclonal antibody production

SummaryWe present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hydridoma cell line that is relatively rare in a population.

Production of hybridomas secreting antibodies to the cornea

Major controversies exist in the literature on the presence of blood group antigens on the endothelial and stromal layers of the cornea, and the importance of major histocompatibility typing for keratoplasty. Antibodies were raised in BALB/C mice against water soluble proteins of corneal epithelium. Following fusion of spleen cells with myeloma cells (Sp2/0-Ag14) hybrid colonies were maintained in HAT selective medium. The supernates of each colony were measured and screened for antibody production by radioimmunoassay. Gel electrophoresis of the antigen showed nine major bands. The antibodies were partially characterized by cross reaction against other soluble corneal fractions.

Primary Herpes Simplex Subepithelial Dendritic Keratitis

A 37-year-old man developed an acute follicular conjunctivitis with preauricular lymphadenopathy believed to be epidemic keratoconjunctivitis. On the eighth day of his disease, subepithelial dendritic opacities developed in the cornea which were not typical of either epidemic keratoconjunctivitis or herpetic keratitis. A diagnosis of primary herpes simplex virus infection was established by positive viral culture and a rise in serum antibody titer to herpes simplex virus. Subepithelial dendritic keratitis as a manifestation of herpes simplex infection of the cornea has not been previously described. The lesions seen in this patient were not reproducible in rabbits and we believe they represent an unusual host response to the virus. This form of herpetic keratoconjunctivitis is extremely difficult to differentiate from epidemic keratoconjunctivitis. Corticosteroids should be used with caution in cases that are not completely typical of epidemic keratoconjunctivitis.

Hybridoma antibodies to insoluble antigens of the corneal epithelium

Human corneal epithelial water insoluble proteins were used to immunize mice. Immune splenocytes were fused with Sp 2/0-Ag14 mouse myeloma cells by 40% PEG. Hybridoma antibodies obtained by somatic cell fusion were tested by radioimmunoassay. Supernatants from antibody positive hybrid colonies were used in immunofluorescence and crossreaction assays to locate and characterize corneal epithelial antigens. At least three distinct epithelial cell antigens were detected, one of which cross-reacts with rabbit corneal epithelial cells.