Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.     

Studies on Pasteurella gallinarum

In two outbreaks of disease in chickens associated with Pasteurella gallinarum infection swollen and inflamed wattles were symptoms. A bacteriological study was carried out of 42 P. gallinarum strains isolated from the two outbreaks and from sporadic cases in fowls. Serotyping by the gel diffusion precipitin test indicated that all five strains from one outbreak and 82% of isolants from 22 sporadic cases belonged to one serotype, antigenically unrelated to Pasteurella multocida. All four isolants from another outbreak were of a different serotype and were antigenically related to P. multocida serotypes 6 and 9. Significant titres of agglutinating antibodies against hyaluronidase-treated P. gallinarum antigens and precipitating antibodies were detected in sera from chickens infected under field conditions and experimentally.     

Immunoglobulin G antibodies toPseudomonas aeruginosa lipopolysaccharides and exotoxin A in patients with cystic fibrosis or bacteremia

IgG antibodies to ninePseudomonas aeruginosa lipopolysaccharides (LPS) and exotoxin A in sera from 11 patients with bacteremia and 51 patients with cystic fibrosis (CF) were analyzed. The methods used were enzyme immunoassay (EIA) and immunoblotting. Nine of the 11 bacteremic patients were infected with strains expressing an LPS serotype identical to one of the test antigens. In sera from six of these nine patients, antibody homologous to the serotype of the infecting strain was observed. An antibody response to heterologousPseudomonas aeruginosa LPS antigens was observed in nine patients. Eight of the bacteremic patients mounted an antibody response to exotoxin A. Thirty-five CF patients chronically colonized withPseudomonas aeruginosa possessed significantly higher levels of antibody to all of the test antigens than 16 patients with intermittent or no colonization (p<0.001). For exotoxin A and serotype 3 the sensitivity was 91 % and 94 %, and the specificity 94 % and 88 % respectively. When the results for exotoxin A and serotype 3 were combined, the sensitivity was 91 % while the specificity was 81 %. The pronounced antibody response to heterologous LPS antigens, as measured by the EIA and immunoblot, suggests expression of a common antigen determinant. A simplified serological assay utilizing exotoxin A and serotype 3 as test antigens may be useful for detectingPseudomonas aeruginosa infections in patients with CF and chronic colonization and in bacteremic patients from whom cultures are not available.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serotype 2.

A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes of A. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work.     

Comparison of seven commercial kits for detection of antibodies toBorrelia burgdorferi

Five enzyme immunoassay (EIA) and two Western blot (WB) commercial kits were compared for their ability to detect antibodies toBorrelia burgdorferi. The panel of 53 test sera consisted of 25 sera positive for antibodies toBorrelia burgdorferi, 15 sera negative for such antibodies, 5 sera reactive in serologic tests for syphilis, and 8 sera containing antinuclear antibodies and/or rheumatoid factor. The rate of agreement with reference results was 93 %, 90 %, 90 % and 88 % for EIA kits from Diamedix, Cambridge Biotech, Mardx and Sigma respectively. The sensitivity and specificity was 84 % and 100 % respectively for Cambridge Biotech, 76 % and 94 % for Diamedix, 68 % and 83 % for Mardx, and 68 % and 83 % for Sigma. The three confirmatory tests, Cambridge Biotech WB, General Biometrics P39 EIA and Mardx WB, demonstrated 75 %, 60 % and 63 % agreement respectively. The sensitivity and specificity was 52 % and 100 % respectively for Cambridge Biotech WB, 24 % and 100 % for General Biometrics P39 EIA, and 44 % and 100 % for Mardx WB. The results demonstrate the variable performance of commercial serologic kits for detection of antibodies toBorrelia burgdorferi. WB appears to be a better confirmatory test than the single protein EIA.     

The outer membrane of Pasteurella multocida 3:A protects rabbits against homologous challenge.

The protective efficacy of a vaccine purified from the Pasteurella multocida 3:A outer membrane (OM) was evaluated in rabbits by homologous challenge. Twenty-seven rabbits were divided into four groups: 1, vaccinated with OM and challenged; 2, nonvaccinated and challenged; 3, vaccinated with OM only; and 4, nonvaccinated and not challenged. Rabbits were immunized intranasally with 1 mg of OM protein on days 0, 7, 14, and 35, challenged intranasally on day 49, and killed on day 63. Mortality rates were 0, 67, 0, and 0% for groups 1 through 4, respectively. The prevalence of pneumonia was reduced from 73 (group 2) to 20% (group 1). The severity of pneumonia was reduced from 0.62 (group 2) to 0.07 (group 1), as measured by the group lesion index. The number of P. multocida in nasal cavities was reduced from 3.89 x 10(5) (group 2) to 6.19 x 10(2) (group 1). The geometric mean number of P. multocida in lungs was 8,360,000-fold less in group 1 than in group 2. Similarly, the prevalence of P. multocida colonization in nonrespiratory organs was reduced from 47 (group 2) to 4% (group 1). Furthermore, group 1 and 3 rabbits developed significantly elevated immunoglobulin A antibodies in nasal secretions and lung lavages and significantly elevated immunoglobulin G antibodies in lung lavages and sera. In addition, rabbit immune sera contained antibodies against P. multocida OM proteins and lipopolysaccharides and inhibited P. multocida proliferation in mouse lungs. These results indicate that a vaccine prepared from the OM of P. multocida provides a significant protection in rabbits against homologous challenge.     

Pasteurella multocida and Bordetella bronchiseptica infections in rabbits.

The natural history of infection with Pasteurella multocida and Bordetella bronchiseptica in domestic rabbits was studied prospectively at a commercial rabbitry. At weaning, about 25% of rabbits had nasal infections with P. multocida and 75% had infections with B. bronchiseptica. Infection of weanling rabbits paralleled nasal infections of their dams. The proportion of rabbits with both infections increased with age. At 2 to 4 months old, about 50% of rabbits with P. multocida or P. multocida and B. bronchiseptica infections had upper respiratory disease (URD), whereas rabbits with B. bronchiseptica infection had no disease. In rabbits about 10 months old, 75% with P. multocida or P. multocida and B. bronchiseptica infections had URD, whereas virtually none with B. bronchiseptica infection had disease. Disease of the nares, paranasal sinuses, middle ears, and lungs was associated with P. multocida and not B. bronchiseptica infection. In adult rabbits with nasal P. multocida infection, with or without signs of URD, about 80% had concurrent infection of the paranasal sinuses and middle ears and 20% had infection of the bronchi and lungs. In rabbits without nasal P. multocida infection, 20 to 35% had P. multocida infection of the paranasal sinuses and middle ears. Weanling rabbits with and without P. multocida infection had similar immunoglobulin G (IgG) levels. In rabbits observed prospectively, the only antibody differences between those transiently and persistently infected with P. multocida were a diminished IgA response in nasal lavages and an earlier IgM response in sera of transiently infected rabbits. IgG levels increased with the duration of infection. There was no relationship between immunoglobulin levels and freedom from P. multocida infection.     

Antibody responses to Campylobacter infections determined by an enzyme-linked immunosorbent assay: 2-year follow-up study of 210 patients

An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.     

Comparison of six different commercial IgG-ELISA kits for the detection of TBEV-antibodies

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a pathogenic human flavivirus endemic in some parts of Europe and Asia. Commercial enzyme immunoassays (EIA) for the detection of IgG antibodies are often used in TBEV-seroprevalence studies, as well as for the confirmation of a successful vaccination against TBEV. However, the detection of TBEV-specific antibodies can be biased by the cross-reactivity between different flavivirus genera. OBJECTIVES: To compare different EIA test systems for the detection of TBEV-IgG antibodies. STUDY DESIGN: Six commercial EIA kits for the detection of TBEV-specific antibodies are compared, using serum panels (n=139) of subjects with a documented clinical history (109 sera from TBEV infected patients, 30 sera from people vaccinated against TBEV). For the analysis of possible cross-reactivities, 24 sera from yellow fever vaccinated people and 13 sera positive for Dengue virus-specific antibodies were also included. RESULTS: The sensitivity of the different TBEV test systems ranges from 73 to 99%. However, when testing the yellow fever and Dengue virus positive specimens, problems with the flavivirus cross- reactivity become obvious, resulting in specificities between 14 and 81%. CONCLUSIONS: This study shows the necessity of further improvement of the existing TBEV test systems regarding both sensitivity and specificity.     

Hyperimmune serum from rabbits immunized with potassium thiocyanate extract of Pasteurella multocida protects against homologous challenge.

Hyperimmune rabbit sera directed to the KSCN extract of 3:A Pasteurella multocida were characterized by enzyme-linked immunosorbent assay (ELISA), presolubilized cell radioimmunoprecipitation, and immunoblotting analysis. The results showed that the hyperimmune serum had a very high titer of immunoglobulin G ELISA antibody and a negligible immunoglobulin A ELISA antibody, precipitated 10 different outer membrane protein antigens by radioimmunoprecipitation, and reacted to 10 different membrane vesicle antigens of P. multocida by immunoblotting analysis. The hyperimmune rabbit sera were also evaluated for protective efficacy against experimental rabbit pasteurellosis by homologous challenge. Thirty-six rabbits were divided into four groups. Group 1, 2, and 3 rabbits were inoculated intranasally with hyperimmune rabbit serum, phosphate-buffered saline, or normal rabbit serum, respectively, at 24 h prior to and 24, 48, and 72 h after intranasal challenge with the virulent homologous P. multocida strain. Group 4 rabbits were inoculated with normal rabbit serum without challenge. Necropsies of surviving rabbits were performed 2 weeks postinfection. The mortality rates for groups 1 through 4 were 25% (3 of 12), 67% (8 of 12), 75% (6 of 8), and 0% (0 of 4), respectively. The prevalence and severity of pneumonia were significantly lower in the hyperimmune serum-treated rabbits. The prevalence of P. multocida colonization in lungs was significantly lower in group 1 rabbits, and the geometric mean CFU of P. multocida in lungs was 59,166-fold less in group 1 rabbits than in group 3 rabbits. The geometric mean CFU of P. multocida in nasal cavities of group 1 rabbits was significantly lower than that of group 3 rabbits. All challenged rabbits (groups 1,2, and 3) had elevated nasal immunoglobulin A and pulmonary (lung lavage) immunoglobulin A antibody levels at necropsy (day 14 postinfection). Similarly, all challenged rabbits had elevated levels of ELISA immunoglobulin G antibody in serum at day 14 but not at day 7 postinfection, indicating that rabbits receiving hyperimmune serum can mount a specific humoral immune response against the homologous challenge P. multocida organisms. We concluded that hyperimmune serum directed to the KSCN extract of 3:A P. multocida provides significant protection against homologous challenge in rabbits.     

Dot-enzyme immunoassay for visual detection of antibodies to pseudorabies virus in swine serum.

A modified solid-phase enzyme immunoassay (EIA) is described for the visual detection of anti-pseudorabies virus (anti-PRV) antibody in porcine serum. Dots of PRV antigens were adsorbed to nitrocellulose paper (hence the name dot-EIA), and the remaining nonspecifically reactive sites were blocked with bovine serum albumin or skim milk powder. After immersion in test serum, bound antibodies were reacted with a peroxidase-conjugated anti-porcine immunoglobulin G (H & L). Positive reactions were easily visualized as brown dots after enzyme degradation of a substrate containing hydrogen peroxide and diaminobenzidine. The dot-EIA was comparable to the serum neutralization test and the standard microtiter EIA in its ability to detect antibody in the sera of pigs 9 days after experimental infection and 12 days after contact with infected pigs. The sensitivity and specificity of the dot-EIA relative to the serum neutralization test and the standard EIA were determined from the testing of 856 field sera from the United Kingdom, the United States, and Canada. In all comparisons, both the relative sensitivity and specificity of the dot-EIA were in the order of 98 to 99%. The dot EIA appears to have potential application as a rapid and economical field test in the diagnosis of PRV infection.     

Serum-specific antibodies in rabbits naturally infected with Trichophyton mentagrophytes.

In the present study, we investigated the humoral immune response of rabbits to Trichophyton mentagrophytes (sensu lato) proteins obtained from keratin-rich media in vitro. The test rabbits were naturally infected with T. mentagrophytes. The production of keratinolytic enzymes in T. mentagrophytes was stimulated by growing cultures with keratin as a sole nitrogen source. The proteins were isolated from a protein extract prepared from the fungal mat. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed three bands. Bands with Mr 20 and 30 kDa were glycosylated, whereas a band of 18 kDa was not. The rabbits' humoral immune responses to the total protein extract of T. mentagrophytes and to the proteins with keratinolytic activity was studied by immunoblotting. IgG from infected rabbits' sera revealed eight dominant bands with apparent molecular weights between 20 and 75 kDa. Bands of 20, 30 and 33 kDa appeared with a frequency rate of 76% only on immunoblots of infected rabbit serum. Using an indirect enzyme-linked immunosorbent assay (ELISA), we observed a significant increase in specific antibodies in a group of infected rabbits compared to a control group (P < 0.001). The ELISA exhibited 95% sensitivity and 83% specificity at the optimal cut-off value, with 90% predictive values of a positive and a negative result. Under these conditions, it could be used in the accurate detection of specific antibodies in sera of infected rabbits.     

Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

Specificities and Opsonophagocytic Activities of Antibodies to Pneumococcal Capsular Polysaccharides in Sera of Unimmunized Young Children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits.

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.     

Western immunoblotting with five Treponema pallidum recombinant antigens for serologic diagnosis of syphilis

Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.     

A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis.

Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb.     

斑点ELISA与环幼沉淀试验诊断旋毛虫病的研究

目的比较斑点ELISA法（Dot—ELISA）和玻片环幼沉淀试验（CPT）检测感染旋毛虫大鼠血清抗体的敏感性和特异性。方法采用Dot-ELISA和CPT两种免疫学诊断技术检测实验感染旋毛虫大鼠血清特异性抗体。结果Dot．ELISA和CPT法检测阳性率分别为97．5％和95．O％,差异无统计学意义（x2=0．3463,P〉0．05）。同时用此两种血清学方法检测正常大鼠、斯氏狸殖吸虫感染大鼠、日本血吸虫感染兔及蛔虫病人血清,除1例蛔虫病人CPT阳性外,其他均为阴性。从第2周开始旋毛虫感染大鼠Dot—ELISA和CPT均能测出抗体,第5周达高峰。结论Dot．ELISA和CPT对旋毛虫特异性IgG抗体的检测均有较好的敏感性和特异性。     

A multi-laboratory evaluation of an enzyme-linked immunoassay quantitating human antibodies to Streptococcus pneumoniae polysaccharides

An enzyme-linked immunoassay (EIA) is described and evaluated which quantitates human antibodies to serotype specific S. pneumoniae polysaccharide (PnPs) in human sera. Based on the observations previously described by Koskela [1], native PnPs are used as coating antigens and sera are absorbed with a soluble pneumococcal absorbant material containing C-polysaccharide (CPs) to ensure measurement of serotype specific anti-PnPs antibodies. The robustness of this method was evaluated by ten laboratories using the same reagents, protocol, and five human serum samples. Reproducible antibody values were obtained for IgM, IgG, and IgA antibodies to five different PnPs serotypes, 3, 6B, 14, 19F, and 23F. The overall mean percent coefficients of variation in this interlaboratory study for all five serotype specific anti-PnPs determinations with the five coded sera were 30% for IgG, 37% for IgM, and 36% for IgA. This assay can be standardized for quantitation of serotype specific anti-PnPs antibodies, allowing comparison of antibody values in vaccine trials evaluating pneumococcal vaccines.     

Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice.

The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 10(6) CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P less than 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P less than 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P greater than 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs.