Measurement of fecal lactoferrin as a marker of fecal leukocytes.

While diarrheal illnesses are extremely common in communities and hospitals throughout the world, an etiologic diagnosis may be expensive and cost-ineffective. Although the presence of fecal leukocytes are helpful in the diagnosis and specific therapy of inflammatory diarrheas, this requires prompt microscopic examination of fecal specimens (preferably obtained in a cup rather than a swab or diaper) by a trained observer. We developed a simple, sensitive test for the detection of leukocytes in fecal specimens using antilactoferrin antibody. Whereas radial immunodiffusion detected 0.02 micrograms of lactoferrin (LF) per microliter or greater than or equal to 2,000 leukocytes per microliter, latex agglutination (LA) readily detected greater than or equal to 0.001 micrograms of LF per microliter or greater than or equal to 200 leukocytes per microliter added to stool specimens. Despite the destruction or loss of morphologic leukocytes on storage for 1 to 7 days at 4 degrees C or placement of specimens on swabs, measurable LF remained stable. Initial studies of stool specimens from six patients with Salmonella or Clostridium difficile enteritis were positive and those from three controls were negative for LF by LA. Of 17 children in Brazil with inflammatory diarrhea (greater than or equal to 1 leukocyte per high-power field), 16 (94%) had LF titers of greater than 1:50 by LA, whereas only 3 of 12 fecal specimens with less than 1 leukocyte per high-power field on methylene blue examination and none of 7 normal control specimens had an LF titer of greater than 1:50 by LA. Of 16 fecal specimens from patients with C. difficile diarrhea (cytotoxin titers, >/= 1:1,000), 95% (n = 15) had detectable LF by LA (in titers of 1:100 to 1: 800). Finally, of 48 fecal specimens from healthy adult U.S. volunteers before and after experimental shigellosis and of 29 fecal specimens from children with documented shigellosis and hospitalized controls in northeastern Brazil, fecal LF titers ranged from 1:200 to >/= 1:5,000 in 96% (25 of 26) samples from patients with shigellosis (and reported positive for fecal leukocytes), while 51 controls consistently had fecal LF titers of 相似文献   

Immunity to Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strains represent the most frequent etiological agent of travelers diarrhea. Challenge studies with several of these strains were undertaken in volunteers to evaluate the mechanisms of disease-induced immunity. Seventeen students and other community volunteers were given 10(6) or 10(8) organisms of E. coli B7A (O148:H28), which produces heat-labile and heat-stable enterotoxins. Ten individuals developed diarrheal illness closely resembling natural travelers diarrhea; of these ten, rises in titer of serum antitoxin and anti-O antibody occurred in eight (80%). Eight of the volunteers who developed diarrhea in the first test agreed to undergo rechallenge 9 weeks later with 10(8) B7A organisms. Only one of these eight "veterans" developed diarrhea versus seven of twelve controls given the same challenge (P = 0.05). Despite clinical protection, all "veterans" excreted B7A after rechallenge. Four controls who developed diarrhea during the homologous B7A rechallenge test were rechallenged 9 weeks later with 10(9) organisms of E. coli strain E2528-C1 (O25:H-), which produces only heat-labile enterotoxin and possesses a different O, H, and pili antigen composition than B7A. Three of four "veterans" and two of six controls developed comparable diarrhea. These studies demonstrate that prior disease due to enterotoxigenic E. coli confers homologous immunity against subsequent challenge, and the operative mechanism apparently is not bactericidal and is not mediated by serum anti-O antibodies. Heterologous protection was not conferred where the only common antigen was heat-labile enterotoxin, indicating that serum infection-derived antitoxin to heat-labile enterotoxin also is not protective.     

Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n = 45; 3 to 10 per day, n = 97; >10 per day, n = 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r = 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.     

Prospective multicenter evaluation of a new immunoassay and real-time PCR for rapid diagnosis of Clostridium difficile-associated diarrhea in hospitalized patients

In a prospective multicenter study, 367 fecal samples from 300 patients with diarrhea were tested for Clostridium difficile-associated diarrhea (CDAD) with a new immunochromatography assay for toxins A and B (ICTAB), a real-time PCR on the toxin B gene, and the cell cytotoxicity assay. Twenty-three (6.2%) of the 367 fecal samples were positive by the cell cytotoxicity assay. With the cell cytotoxicity assay as the "gold standard," the sensitivity, specificity, positive predictive value, and negative predictive value for the ICTAB assay and real-time PCR were 91, 97, 70, and 99%, and 87, 96, 57 and 99%, respectively. In conclusion, both the ICTAB and the real-time PCR can be implemented as rapid screening methods for patients suspected of having CDAD.     

Kinetics of local and systemic immune responses after vaginal immunization with recombinant cholera toxin B subunit in humans

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.     

Kinetics of Local and Systemic Immune Responses after Vaginal Immunization with Recombinant Cholera Toxin B Subunit in Humans

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.     

Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection

Clostridium difficile causes antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treat C. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequent C. difficile reinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.     

Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD) occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF) may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P<0.05) in C. difficile challenged groups compared to unchallenged controls, but insignificant (P>0.05) in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.     

Antibacterial and Antitoxin Responses in the Serum and Milk of Cholera Patients

Antibacterial and antitoxin responses in the acute and convalescent (7 to 10 days) sera of 14 cholera patients were determined by various serological techniques. Similar studies were also carried out with corresponding milk samples of six of these patients who were lactating women. A significant rise in antibacterial titers was observed in all convalescent serum and milk samples. A similar rise in antitoxin titers was observable in all serum and four milk samples. Specificity of the antibacterial titers was further evaluated by the indirect hemagglutination test using lipopolysaccharide antigen, and close correlations were noted between these titers and vibrio agglutination (P<0.001) and vibriocidal (P<0.001) titers of sera. Serum and milk convalescent cholera patients could effectively neutralize cholera toxin action in vivo, although the neutralizing activity of serum was higher than that of milk. Determination of antibody titers by the enzyme-linked immunosorbent assay demonstrated that anti-lipopolysaccharide activity in sera belonged predominantly to immunoglobulin M (IgM) and, to a lesser extent, to IgG and IgA, whereas such activity in milk was mostly contributed by secretory IgA, although some IgM antibodies also could be detected. On the other hand, antitoxic activity in convalescent sera primarily belonged to IgG, whereas such activity in milk was almost exclusively contributed by secretory IgA. These results demonstrate that an antibody response in the mammary gland was stimulated due to the antigen exposure in the gut and are consistent with the idea of a common homing pattern of immunocytes within the secretory immune system. Moreover, some differences in the antibody production mechanism between the systemic and secretory immune systems are indicated.     

Factors associated with Clostridium difficile diarrhea in a hospital in Beijing,China

Clostridium difficile is the most common cause of hospital-acquired diarrhea in patients treated with antibiotics, chemotherapeutic agents, and other drugs that alter the normal equilibrium of the intestinal flora. A better understanding of the risk factors for C. difficile-associated disease (CDAD) could be used to reduce the incidence of CDAD and the costs associated with its treatment. The aim of this study was to identify the risk factors for CDAD in a cohort of Chinese patients in a Beijing hospital. Medical charts of a total of 130 inpatients (62 males and 68 females) with hospital-acquired diarrhea (45 with CDAD; 85 without CDAD) were retrospectively reviewed. C. difficile toxins A and B were detected in fecal samples using enzyme-linked fluorescence assays. The drugs used by patients with and without CDAD before the onset of diarrhea were compared. Factors that differed significantly between the two groups by univariate analysis were analyzed by multivariate analysis using a logistic regression model. Multivariate analysis showed that cephalosporin treatment was associated with a significantly higher risk of CDAD in hospitalized patients, while treatment with glycopeptides was significantly associated with a reduction in CDAD (P<0.001 for cephalosporin; P=0.013 for glycopeptides). Our data confirmed previous findings that empirical treatment with cephalosporins is positively associated with CDAD compared to individuals using other CDAD-related drugs. Additionally, we showed that treatment with glycopeptides was negatively associated with CDAD, compared to individuals using other CDAD-related drugs.     

Development of serum and intestinal antibody response to rotavirus after naturally acquired rotavirus infection in man

The temporal characteristics of the response of rotavirus specific IgM, IgG, IgA in serum and secretory antibody in feces to rotavirus were studied in 77 hospitalized patients with rotavirus induced gastroenteritis. The response in serum was characterized by the sequential appearance of rotavirus specific IgM, IgG, and IgA antibody. The IgM antibody appeared to be higher in the acute phase of the disease and was subsequently replaced by the IgG and IgA antibodies. However, the titers of IgG rotavirus antibody in convalescent specimens of serum were found to be statistically significantly lower in patients with severe or prolonged rotavirus infection than in specimens from subjects with mild or moderate disease. Most fecal specimens collected during both the acute and convalescent phase of illness contained virus specific secretory IgA. Higher concentrations of antibody were measured in convalescent samples from patients with prolonged diarrhea and virus shedding. These observations suggest a possible relationship between the severity of rotavirus infection and the nature of systemic and secretory antibody response.     

Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A

BACKGROUND: Clostridium difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis. We studied whether antibody responses to C. difficile toxins affect the risks of colonization, diarrhea, and asymptomatic carriage. METHODS: We prospectively studied C. difficile infections in hospitalized patients who were receiving antibiotics. Serial stool samples were tested for C. difficile colonization by cytotoxin assay and culture. Serum antibody (IgA, IgG, and IgM) levels and fecal antibody (IgA and IgG) levels against C. difficile toxin A, toxin B, and nontoxin antigens were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 271 patients, 37 (14 percent) were colonized with C. difficile at the time of admission, 18 of whom were asymptomatic carriers. An additional 47 patients (17 percent) became infected in the hospital, 19 of whom remained asymptomatic. The baseline antibody levels were similar in the patients who later became colonized and those who did not. After colonization, those who became asymptomatic carriers had significantly greater increases in serum levels of IgG antibody against toxin A than did the patients in whom C. difficile diarrhea developed (P<0.001). The adjusted odds ratio for diarrhea was 48.0 (95 percent confidence interval, 3.4 to 678) among patients with colonization who had a serum level of IgG antibody against toxin A of 3.00 ELISA units or less, as compared with patients with colonization who had a level of more than 3.00 ELISA units. CONCLUSIONS: We find no evidence of immune protection against colonization by C. difficile. However, after colonization there is an association between a systemic anamnestic response to toxin A, as evidenced by increased serum levels of IgG antibody against toxin A, and asymptomatic carriage of C. difficile.     

Serum antibody response to filamentous hemagglutinin in patients with clinical pertussis measured by an enzyme-linked immunosorbent assay

Titers of antibodies to filamentous hemagglutinin (FHA) were determined by enzymelinked immunosorbent assay in acute and convalescent phase serum samples from 158 patients with clinical symptoms typical of whooping-cough. In 96 of the patients the diagnosis was verified by culture. Significant changes in serum levels of IgG, IgM and/or IgA antibodies against FHA were demonstrated in 126 patients (80%). Thus, demonstration of significant changes in FHA antibody titers in serum can be used for serological diagnosis of pertussis. The results also show that high levels of IgG, IgM and/or IgA antibodies in a single serum sample suggest current pertussis infection, but if the diagnosis is based on determinations of FHA antibody titers in a single serum sample the sensitivity is low. The levels of antibody to FHA were compared with previously determined levels of antibodies to pertussis toxin. A significant antibody response against both FHA and pertussis toxin was seen in 111 patients (70 %) while 147 patients (93 %) developed a significant increase in antibodies against one or both antigens.     

To culture or not to culture: fecal lactoferrin screening for inflammatory bacterial diarrhea.

Because of its low yield in unselected specimens, stool culture is often cost ineffective. We tested 55 fecal samples from Fairfax Hospital (46 patients with diarrhea and 9 from controls without diarrhea) for lactoferrin by latex agglutination (LFLA) with the Leukotest (Techlab, Blacksburg, Va.) as a marker for inflammatory diarrhea. Of the 28 samples with Salmonella, Shigella, or Campylobacter infection, 93% had detectable fecal lactoferrin at > or = 1:50 (61% had LFLA titers of > or = 1:400), while 83% of 18 samples with rotavirus or no detectable pathogen were LFLA negative at a titer of 1:50 (100% were negative at 1:400). All 9 controls without diarrhea were LFLA negative at 1:50. The use of fecal lactoferrin to screen for inflammatory diarrhea selects specimens for which stool culture is fivefold more likely to yield an invasive bacterial pathogen (reducing the cost per positive result by over $800) and thus may greatly enhance a cost-effective approach to evaluating diarrheal illness.     

Protection against experimental cholera by oral or parenteral immunization.

Comparisons were made between the antigenic potency and protective capacity of several cholera toxin derivatives. Rabbits were immunized parenterally with 50 microgram of cholera toxin, A subunit, B subunit, procholeragenoid, or Wyeth glutaraldehyde toxoid 20101. Examination of the antibody response curves revealed that cholera toxin elicited serum antitoxin responses that rose more quickly than in the subunit-immunized animals; however, antitoxin levels were of the same magnitude after 10 weeks. Parenteral immunization with procholeragenoid evoked antibody titers that were similar to the toxin, whereas Wyeth toxoid yielded only one-tenth the level of antitoxin. Oral immunization with procholeragenoid as well as Wyeth toxoid resulted in lower serum antitoxin titers than that achieved with parenteral immunization, despite the oral administration of 10 times the parenteral dose. Analysis of protection against live-cell challenge revealed that parenteral administration of procholeragenoid provided the best protection against fluid accumulation. Oral immunization with procholeragenoid also was very effective, whereas oral immunization with B subunit or Wyeth toxoid resulted in minimal protection. Also, the A subunit provided surprisingly more protection than did cholera toxin.     

Comparative analysis of serum antibody responses to Pseudomonas aeruginosa exotoxin A by cystic fibrosis and intensive care unit patients

Pulmonary infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa toxin is one of several proposed virulence factors which may be responsible for chronic P. aeruginosa infections in these patients. With a highly specific, sensitive, and quantitative radioimmunoassay (RIA) and a cell culture assay, the humoral immune responses of CF patients in terms of total antitoxin, antitoxin immunoglobulins A and M, and neutralizing antitoxin were compared with those of P. aeruginosa-infected intensive care unit patients and controls. The P. aeruginosa-infected CF patients were divided into severe and moderate disease groups based on mortality observed over an 8-year period. The intensive care unit patients were divided by the site of infection and the controls were healthy children and uninfected CF patients. Antibodies to toxin were found in the sera of all subjects by radioimmunoassay. Neutralizing antibody was associated with current infection. Elevated titers of antitoxin immunoglobulin A were found only in subjects with pulmonary P. aeruginosa infections. No significant differences in any antibody class were observed between the severe and moderate disease groups. In addition, no differences were observed in the antitoxin immune response of chronically infected CF patients and intensive care unit patients with acute pulmonary infections.     

Arousal of mucosal secretory immunoglobulin A antitoxin in rats immunized with Escherichia coli heat-labile enterotoxin.

Specific serum and mucosal antitoxin levels were determined by enzyme-linked immunosorbent assays in rats immunized with Escherichia coli heat-labile enterotoxin (LT). Immunization by means of a parenteral prime followed by peroral boosts was the only approach that aroused titers of both serum immunoglobulin G (IgG) antitoxin and mucosal secretory IgA antitoxin that were increased fourfold or more over control values. Primary parenteral immunization was effective when given either intraperitoneally or subcutaneously with either Freund complete adjuvant or alum as the adjuvant. The magnitude of the nucosal secretory IgA antitoxin response and the degree of protection against challenge with either LT or viable LT-producing organisms were related to the number and dosage of peroral boosts. LT antigenicity, as determined by enzyme-linked immunosorbent assay, was progressively reduced by toxoiding it with increasing amounts of glutaraldehyde or a carbodiimide; when LT antigenicity was reduced by greater than 50%, the effectiveness of the toxoid in stimulating mucosal antitoxin and providing protection was compromised. Strong protection extended for more than 6 weeks only in rats immunized with a sufficient peroral dosage of LT to arouse mucosal secretory IgA antitoxin titers at least fourfold greater than those of controls. These observations indicate that the ability of LT to stimulate a mucosal secretory IgA antitoxin response is dependent on the antigenicity, route, and dosage of this immunogen; they suggest that the duration of protection in animals immunized by the peroral route is related to the extent of arousal of mucosal secretory IgA antitoxin.     

Prospective study of serum antibodies to Pseudomonas aeruginosa exoproteins in cystic fibrosis.

Serum immunoglobulin G to four purified antigens from Pseudomonas aeruginosa, phospholipase C, alkaline protease, exotoxin A, and elastase, were determined in 62 patients with cystic fibrosis by enzyme-linked immunosorbent assay. The patients were followed for 12 to 24 months in a prospective study. Increased titers, i.e., titers more than 2 standard deviations above those of normal controls, were exclusively found in patients chronically colonized with P. aeruginosa and not in patients harboring only Staphylococcus aureus. The frequencies of elevated titers of antibody to the different antigens varied from 100% (phospholipase C) to 58% (alkaline protease and exotoxin A) to 15% (elastase) in the chronically colonized patients. Mean serum titer levels, expressed as multiples of the age-correlated upper normal limit (=1), were significantly higher to phospholipase C in patients with dual colonization with P. aeruginosa and S. aureus than in those colonized only with P. aeruginosa (P less than 0.001). Conversely, the other three antigens showed significantly higher serum antibody titer levels in patients harboring only P. aeruginosa (P less than 0.001). In five patients who became colonized with P. aeruginosa during the study period, serum antibodies to phospholipase C and exotoxin A increased first. Exceptions to the general pattern of antibody responses were found in three patients chronically colonized with Escherichia coli. They showed a delayed enhancement of anti-phospholipase C titers after the chronic P. aeruginosa colonization. Serum titers were not influenced by exacerbations of pulmonary infection or by antimicrobial therapy. The determination of titers of serum antibody to phospholipase C seems to be a valuable indicator of a chronic colonization with P. aeruginosa. The results further suggest that bacterial metabolism and interactions may influence the antibody response.     

Immunoglobulin G subclass antibodies to measles virus in patients with subacute sclerosing panencephalitis or multiple sclerosis

Mouse monoclonal antibodies specific for human immunoglobulin G (IgG) subclasses and a sensitive immunoassay were used to evaluate the IgG subclass antibody response to measles virus antigens in cerebrospinal fluid and serum samples from 20 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS), and 11 controls with high measles virus antibody titers in serum. In patients with SSPE, measles virus-specific antibodies were found mainly in the IgG1 subclass and the IgG subclass distribution remained unchanged, irrespective of the clinical stage or duration of the disease. In patients with MS and in controls, measles virus activity was also associated mainly with IgG1. However, the activity was significantly lower than that found in patients with SSPE. The results suggest that there is no primary abnormality in humoral immune response to measles virus in patients with MS. The disproportionately high levels of the measles virus-specific IgG1 subclass found in patients with SSPE may be due to persistent antigenic stimulation or reflect a defect in immunoregulatory mechanisms in response to viral infection.     

Detection of tetanus antitoxin using Eu(3+)-labeled anti-human immunoglobulin G monoclonal antibodies in a time-resolved fluorescence immunoassay.

Tetanus antitoxin in human sera was detected with solid-phase immunoassays in microtitration modules coated with tetanus toxoid by using Eu(3+)-labeled anti-human monoclonal antibodies on the basis of an exactly calibrated antibody standard. The use of a time-resolved fluorescence immunoassay (TR-FIA) significantly improved the quantitative detection of tetanus antitoxin over that of the enzyme-linked immunosorbent assay (ELISA) technique because of its high sensitivity and its wide measurement range, detecting antibody levels between 0.001 and 12.5 IU/ml with a single serum dilution of 1:100. For the same purpose, two different serum dilutions (1:100 and 1:1,000) were needed in the ELISA technique. TR-FIA is reproducible and can be performed in 3.5 h. A study of 2,630 serum samples was undertaken to examine the age-dependent distribution of titer levels, indicating the decline of sufficient protection in patients older than 60 years. The wide measurement range of TR-FIA enabled fast examination of large numbers of serum samples without the need for repetition, with further sample dilution, as was often necessary in the ELISA procedure.