Clinical laboratory applications of monoclonal antibodies.

Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories.

Toxicity and pharmacokinetics of cefepime (BMY-28142) following intravitreal injection in pigmented rabbit eyes.

We delineated the dose- and time-dependent retinal toxicity of cefepime (BMY-28142), a new third generation cephalosporin, using electroretinography in pigmented rabbit eyes. Toxicity was evaluated following intravitreal doses ranging from 0.5 to 20mg/0.1ml (N = 18). Electroretinographic patterns at one and two weeks indicated a toxic response to 20 mg of cefepime. B-waves were normal at one and two weeks for rabbits receiving doses of 0.5 to 10mg. Pharmacokinetic analysis after single intravitreal injection of 1 mg of cefepime (N = 3 rabbits/dose) disclosed the following vitreous fluid levels (ug/ml): 645 at Oh, 431 at 8h, 235 at 24h and 23 at 72h. Peak aqueous humor levels (56 ug/ml) were observed at 8h after injection. At 72h, ug/ml was detected in the aqueous fluid.

Synergism between Bacteroides fragilis and Staphylococcus aureus in experimental tibial osteomyelitis.

We explored the potential role of microbial synergy in an experimental rat osteomyelitis model. Osteomyelitis was assessed by gross pathologic conditions and quantitative cultivation of rat tibiae for the implanted organisms 21 days after challenge. When Staphylococcus aureus was used alone, the 50% infective dose (ID50) and the 100% infective dose (ID100) were 400 and 25,000 colony-forming units (CFU), respectively. When Bacteroides fragilis was inoculated alone, the ID50 was 150,000 organisms, and the ID100 was not confidently determined. Subinfectious numbers of B. fragilis added to the staphylococcal inocula yielded an ID100 as low as 20 staphylococci. Mixed inocula with 20 or 200 staphylococci and increasing numbers of B. fragilis yielded a dose-dependent increase in the number of staphylococci isolated from osteomyelitic tibiae. Multiple linear regression analysis confirmed the two inocula and their interaction to be significantly predictive of the 21-day quantitative assessment of staphylococci (r = 0.80). Synergy was most striking at low bacterial inocula. When even large numbers of S. aureus were added to B. fragilis, the B. fragilis inoculum required to initiate B. fragilis osteomyelitis was essentially unchanged. We conclude that small numbers of B. fragilis allow remarkably low numbers of S. aureus (20 or 200 CFU) to establish osteomyelitis in the rat.

Intravitreal ceftazidime in a rabbit model: dose- and time-dependent toxicity and pharmacokinetic analysis.

We delineated the dose-and time-dependent retinal toxicity of intravitreal ceftazidime using electroretinography (ERG) in phakic rabbit eyes. Toxicity was evaluated following intravitreal doses ranging from 0.5 to 50 mg/0.1 ml. Eyes were examined prior to injection, at one day, and at seven days after injection. ERG patterns at one and seven days indicated a toxic response to doses of 20 to 50 mg. Doses of 10 mg or less gave a transient depression of the B-wave at one day, but a return to normal was observed at the seven-day evaluation. Pharmacokinetic analysis following intravitreal injection of 2 mg of ceftazidime into uninfected phakic rabbit eyes disclosed vitreous levels of: 1711 micrograms/ml at 0 hr; 1340 micrograms/ml at 8 hr; 451 micrograms/ml at 24 hr; 270 micrograms/ml at 48 hr and 67 micrograms/ml at 72 hr. Peak aqueous humor level was 139 micrograms/ml 8 hr post-injection. Potentially therapeutic drug levels were obtained in the aqueous and vitreous humors following a nontoxic intravitreal dose of ceftazidime in a rabbit model.

Subconjunctival administration of ceftazidime in pigmented rabbit eyes.

The ocular kinetics of ceftazidime, a third-generation cephalosporin, were examined in phakic and aphakic pigmented eyes of rabbits following subconjunctival injection (100 mg). Peak ceftazidime concentrations (mean +/- SE, n = three to five rabbits per determination) were as follows: phakic eyes, 40.2 +/- 7.3 mg/L in aqueous humor and 11.2 +/- 0.6 mg/L in vitreous humor at one hour; aphakic eyes, 30.5 +/- 4.8 mg/L in aqueous humor and 15.8 +/- 2.4 mg/L in vitreous humor at one hour. The ability of ceftazidime to eliminate an incipient bacterial infection was also studied. Ten aphakic rabbits received intravitreal injections of 50 colony-forming units (cfu) of Pseudomonas aeruginosa. Six of the ten immediately received a subconjunctival injection of ceftazidime (100 mg). At 48 hours following injections, four of four control eyes yielded bacterial counts greater than 6.2 X 10(6) cfu/mL. Of the six that received ceftazidime, five were sterile and one yielded 10 cfu/mL.

Multistrain comparison of three antimicrobial prophylaxis regimens in experimental postoperative Pseudomonas endophthalmitis.

We compared subconjunctivally administered ceftazidime and BMY 28142, two third-generation cephalosporins, to a regimen of gentamicin and cefazolin for their ability to prevent experimental Pseudomonas postoperative endophthalmitis in rabbits. After extracapsular lens extraction, an inoculum of Pseudomonas was injected into the vitreous; one of the three antimicrobial regimens was then administered subconjunctivally. All 25 eyes treated with gentamicin and cefazolin become infected (P = 1). Two of 25 eyes treated with BMY 28142 became infected (P less than .001). None of the 25 eyes treated with ceftazidime became infected (P less than .001).

Arachidonic acid facilitates experimental chronic osteomyelitis in rats.

Arachidonic acid was used as a facilitating agent in experimental rat Staphylococcus aureus osteomyelitis and compared with the more commonly used agent, sodium morrhuate. The injection of arachidonic acid or sodium morrhuate and S. aureus into rat tibiae caused increased quantitative bacterial bone counts, gross bone pathology, roentgenographic changes, and weight loss. The doses required to produce these changes appeared to be lower for arachidonic acid.

Model of experimental chronic osteomyelitis in rats.

We describe here a Sprague-Dawley rat model for chronic osteomyelitis. Staphylococcus aureus and sodium morrhuate were implanted by either microdrilling or direct needle injection into the tibiae of rats. Of 107 rats, 87 (81%) developed osteomyelitis when a high-speed drill was used for implantation, and 27 (51%) of 53 rats developed osteomyelitis by direct needle inoculation (chi square = 9.81, P less than 0.01). Demonstrated histopathological changes included the presence of resorption bays filled with osteoclasts. Quantitative microbiological monitoring of tibial count confirmed disease chronicity, yielding stable numbers of CFU (10(6.29 +/- 0.27) ) of S. aureus over 70 days. Infected animals became anemic and lost weight. The erythrocyte sedimentation rates and leukocyte counts were not elevated. Roentgenograms provided the best correlation with the number of organisms in infected tibiae (r2 = 0.80). Rats with infected tibiae were treated with either oxacillin (120 mg/kg per day) or ceftriaxone (50 mg/kg per day). Treatment over 14 or 28 days reduced S. aureus counts in tibiae but did not reliably sterilize infected bones, suggesting that this model was resistant to prolonged antimicrobial therapy.

Effect of inoculum size on the induction of endophthalmitis in aphakic rabbit eyes.

A reproducible animal model is necessary to examine the use of antimicrobial agents for prophylaxis and treatment of bacterial endophthalmitis. We determined the minimum inoculum size of S. aureus and P. aeruginosa that consistently produced endophthalmitis when injected into aphakic rabbit eyes immediately following surgery. Both anterior chamber and intravitreal injections were examined. For S. aureus, an intravitreal inoculum of 19.3 +/- 7.5 CFU and an anterior chamber inoculum of 50.5 +/- 4.0 CFU were required. For P. aeruginosa, an intravitreal inoculum of 5.5 +/- 2.6 CFU and an anterior chamber inoculum of 97.5 +/- 10.7 CFU consistently produced a fulminant infection. Lower inocula of both bacteria produced endophthalmitis in both locations, but the effect was inconsistent.

Pharmacokinetic analysis of intravitreal ceftriaxone in monkeys.

Ocular toxic effects of intravitreally injected ceftriaxone sodium and its rate of clearance after a single dose were studied in rhesus and cynomolgus monkeys. Doses of 2, 2.5, and 3 mg/0.1 mL were injected into the vitreous cavity. Electroretinograms performed 15 minutes, 24 hours, one week, and two weeks after injection were not significantly different from preinjection electroretinograms. Lens opacification occurred in one eye receiving 2.5 mg of ceftriaxone. Histopathologic findings in the retina were normal in all eyes injected intravitreally. Levels of ceftriaxone after a 2-mg intravitreal dose were 609 mg/L at 0 hours, 434 mg/L at 24 hours, and 19 mg/L at 100 hours in the vitreous; and 1 mg/L at 0 hours, 116 mg/L at 24 hours, and 9 mg/L at 100 hours in the aqueous.

Intravitreal ceftriaxone in a rabbit model. Dose- and time-dependent toxic effects and pharmacokinetic analysis.

Ceftriaxone's toxic effects were assessed in albino rabbits after intravitreal injection. Doses up to 5 mg did not alter the B-wave amplitude. Following 7.5-and 20-mg doses, B-wave amplitude ratios were depressed at 24 hours and normal at seven and 14 days. A 50-mg dose caused a temporarily flat B-wave 24 hours after injection. At two weeks this ratio exhibited a moderate increase but remained 2 SDs below the mean preinjection ratio. Eyes receiving up to 20 mg were normal histologically at 14 days. A 50-mg dose induced generalized retinal edema and disruption of the retinal layers 24 hours after injection; at two weeks there were no histologic changes in the retina. Immediately after a 2-mg intravitreal injection, vitreous ceftriaxone levels were 1,345 +/- 4.9 mg/L; by 72 hours they had decreased to 17.6 +/- 1.4 mg/L. The mean peak aqueous level was 80.2 +/- 12.2 mg/L at 72 hours.

Unregulated production of virus and/or sperm specific anti-idiotypic antibodies as a cause of AIDS.

A network of idiotypic and anti-idiotypic antibodies is often suggested as the basis for cellular interactions that maintain a steady-state immunological equilibrium. This hypothesis proposes that repeated exposure to certain external antigens--ie, both viral and sperm--stimulates an unregulated production of a uniquely potent immunomodulating idiotypic antibody(ies). In a genetically predisposed individual, this particular antibody(ies), which is also an autoantibody(ies), results in a cellular immune deficiency. This disruption in the immune system permits opportunistic infection and thus the acquired immune deficiency syndrome. This hypothesis, which is readily testable and which does not involve a primary pathogen, can explain both the active induction of this disease in, as well as its passive transfer to, all at-risk populations.

Detection of specific bacterial antigen in urine of patients infected with Bacteroides fragilis.

An indirect enzyme-linked immunosorbent assay was developed to detect specific Bacteroides fragilis antigen(s) in human urine. Specimens collected within 72 hr of a positive culture were centrifuged, dialyzed, and treated with Tween 20, polyethylene glycol, and bovine serum albumin. Goat hyperimmune gamma-globulin to B. fragilis strain ATCC 23745 was added and incubated, and supernatants were tested for antibody activity to polysaccharide-protein antigen of the same organism. Mean +/- SD results, reported as percentage inhibition of control values and interpreted blindly, were as follows: 29 normal subjects, 9.8% +/- 6.0%; 22 patients with Enterobacteriaceae bacteremia, 6.0% +/- 5.1%; six patients with nonbacteremic infections due to B. fragilis, 22.3% +/- 10.3%; and nine patients with B. fragilis bacteremia, 28.7% +/- 10.2%. Three of six nonbacteremic patients and eight of nine bacteremic patients yielded values greater than 2 SD of control values. None of 22 patients with Enterobacteriaceae bacteremia was falsely positive (specificity, 100%).

Ocular pharmacokinetics of ceftriaxone following subconjunctival injection in rabbits.

Aqueous and vitreous kinetics were studied after anterior subconjunctival injection of 100 mg of ceftriaxone sodium in phakic and aphakic rabbit eyes. Mean peak ceftriaxone concentrations (microgram per milliliter +/- SE, n = 3 to 5 rabbits per determination) were as follows: phakic eyes, 159.5 +/- 42 at one hour in aqueous humor and 25.3 +/- 6.6 at two hours in vitreous fluid; aphakic eyes, 105.1 +/- 20.5 at one hour in aqueous humor and 43.1 +/- 15.4 at one hour in vitreous humor. The ability of ceftriaxone to eliminate an incipient bacterial infection was also evaluated. Ten aphakic rabbits were challenged intravitreally with 700 colony-forming units of Staphylococcus aureus. Six of the ten immediately received subconjunctival injections of ceftriaxone sodium (100 mg). At 48 hours following the challenge, all four control eyes yielded greater than 5.6 X 10(5) colonies per milliliter. In the six eyes receiving ceftriaxone, five were sterile and one yielded 1.4 X 10(2) colonies per milliliter.

Iontophoresis of gentamicin into aphakic rabbit eyes. Sustained vitreal levels.

The authors evaluated the intraocular penetration of gentamicin (50 mg/ml) into aphakic rabbit eyes following anodal iontophoresis (0.75 mA for 10 min). Gentamicin levels were determined at 0.5, 4, 8, 16, and 24 hrs after iontophoresis (n = 6 eyes for each time) using an agar diffusion bioassay. Peak levels of 72.04 +/- 6.1 (means +/- SE) micrograms/ml for the corneas and 77.8 +/- 3.0 micrograms/ml for the aqueous humor were obtained at 30 min after iontophoresis. The peak vitreous level was 10.4 +/- 0.4 micrograms/ml, which was found at 16 hrs after iontophoresis. Therapeutic levels of 6.2 +/- 3.0 micrograms/ml were still present in the vitreous humor 24 hrs after iontophoresis. Iontophoresis appears to be an effective noninvasive method for delivering therapeutic levels of gentamicin into ocular tissues and fluids of the aphakic rabbit eye.

Detection of a specific bacterial antigen in urine of rats with Bacteroides fragilis infection.

Human intra-abdominal infections frequently yield Bacteroides fragilis and require specific antimicrobial and surgical therapy. Noninvasive immunologic assessment of this organism might allow more optimum therapy. Therefore we raised antisera in goats to Bacteroides fragilis ATCC 23745 and allowed it to react with a solid-phase capsular polysaccharide-protein antigen extracted from the same organism. Preliminary work disclosed that 10 ng/ml antigen could be detected in competition assays in both saline and dialyzed rat urine. Results were manifest by diminution of bound antiglobulin alkaline phosphatase conjugate in an antigen-mediated antibody-inhibition enzyme-linked immunosorbent assay. Rats were then infected intra-abdominally with (1) B. fragilis ATCC 23745; (2) one of eight recent clinical isolates of B. fragilis; or (3) one of nine isolates representative of Enterobacteriaceae. Seventy-two rat urine samples obtained prior to infection disclosed essentially no assay inhibition: 98.3% +/- 10.3 (1 S.D.). Mean values of reagent antibody activity after incubation with urine aliquots from 24 hr samples collected between 24 and 72 hr were (1) strain 23745 (n = 35) 70.9% +/- 2.6 (S.E.); (2) eight isolates of B. fragilis (n = 49) 86.8% +/- 1.9; (3) nine isolates of Enterobacteriaceae (n = 47) 100.9% +/- 1.0; and (4) shams (n = 29) 95.5% +/- 1.55. Ascribing values less than or equal to 77.7% (2 S.D.) as positive, seven of the eight clinical B. fragilis isolates causing infection were detected in at least one 24 hr urine sample (sensitivity = 87% by organism); 12 of 17 infected rats were correctly identified as positive by at least one urine (sensitivity = 70.6% by rat). Specificity, as assessed in the Enterobacteriaceae group, was 89% (by organism) and 94.5% (by rat). Collectively, these results suggest the presence of a potentially specific, soluble antigen excreted in the urine of rats with B. fragilis infection.

Bacteria-immune system interactions. IV. Effect of various treatments on the binding of bacteria to human lymphocytes.

Pretreatment of lymphocytes with selected saccharides inhibited the binding of Bacillus globigii (B. globigii) to lymphocytes but had no effect on Escherichia coli 2 (E. coli 2) adherence. The binding of both bacteria was prevented by lymphocyte pretreatment with trypsin or pronase. Periodate treatment of lymphocytes inhibited the binding of E. coli 2 but not of B. globigii. This inhibition could be reversed by borohydrate treatment of lymphocytes. Trypsin, pronase or periodate treatment of bacteria had no effect on the binding to lymphocytes. The data obtained suggest that: (a) E. coli 2 and B. globigii have different mechanisms of binding to lymphocytes; and (b) the lymphocyte receptor for E. coli 2 is of a glycoprotein nature.

Bacteria-immune system interactions. V. Escherichia coli cell-wall components involved in binding to lymphocytes.

Various bacterial cell-wall components were tested for their ability to inhibit Escherichia coli 2 binding to lymphocytes. The binding was inhibited by outer membrane extracts of E. coli 2 but not by similar extracts from E. coli 0. In addition, mannose-binding protein, inner membrane or lipopolysaccharide extracts of E. coli 2 or lipoteichoic acid extracts of Bacillus globigii had no effect on lymphocyte-E. coli 2 interaction. The interaction did not appear to be mediated by fimbriae, since such structures could not be detected by electron microscopy. Comparison of the outer membrane proteins of a binder (E. coli 2) and a nonbinder (E. coli 0) bacteria revealed differences in their number and position.

SJ-GC, a modified complete medium for growth of Neisseria gonorrhoeae.

A broth medium that supports growth of Neisseria gonorrhoeae to high densities with minimal lysis is described. With most gonococcal strains, inocula of 2 x 10(6) colony-forming units per ml yielded greater than 109 colony-forming units per ml after 8 h of incubation. Scale-up cultures produced 5 to 12 g (wet weight) of cells per liter.