IgE-binding and cross-reactivity of a new 41 kDa allergen of codfish

Background:   A 41-kDa IgE-reactive protein (p41) was purified from raw cod extract. This protein is homologous to an aldehyde phosphate dehydrogenase (APDH). The present study aims to evaluate the IgE-binding and the cross-reactivity of this protein in 13 patients allergic to codfish.
Methods:   IgE binding of sera from 13 patients allergic to codfish was tested by Sepharose RIA and by Western blot.
Results:   Among the 13 patients, only 4 had specific IgE to APDH detected by APDH-Sepharose RIA. The two patients who had the highest level of specific IgE to human APDH also had a class 5–6 CAP-RAST IgE level to codfish, but two other patients with a class 5 had a negative APDH-Sepharose IgE-RIA. Relative content of APDH was higher in extracts of commercial nonfrozen fish, compared to pre rigor mortis , post rigor mortis and frozen commercial codfish. A high homology of codfish APDH was found with the corresponding human enzyme. A significant inhibition of APDH-Sepharose by human and, to a lesser extent, by rabbit APDH was observed. Western blot of APDH codfish extract showed two bands at 41 and 36 kDa, respectively.
Conclusions:   We have characterized a new allergen from codfish, which had a high level of homology in different species. The p41 relative content of extracts from nonfrozen codfish was higher than in the other samples assessed.     

Western immunoblot analysis and serologic characterization of Blastomyces dermatitidis yeast form extracellular antigens.

A major 98-kDa extracellular protein antigen of Blastomyces dermatitidis was shown in Western blot (immunoblot) analysis to be highly reactive with serum antibodies from patients with blastomycosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of yeast form B. dermatitidis culture filtrates and cell extracts demonstrated over 50 proteins, only 24 of which were immunoreactive. Of these, a 98-kDa protein was found to be the most specific and was isolated. This protein was found in both broth culture filtrates and extracts of yeast forms. Western blot tests with this antigen detected serum antibodies in 91% of 32 patients with clinically proven blastomycosis, in contrast to an enzyme-linked immunosorbent assay (ELISA) with DEAE-purified antigen, which detected 88% of the cases. The Western blot test also exhibited lower reactivity with a panel of sera from patients with heterologous fungal infections that were cross-reactive in the ELISA with DEAE-purified B. dermatitidis antigen. The 98-kDa protein electroeluted from polyacrylamide gels was identical to the diagnostic A antigen used in the blastomycosis immunodiffusion test. Comparison of the 98-kDa antigen with a previously described 120-kDa yeast form cell wall protein antigen of B. dermatitidis showed that these two antigens are almost identical.     

Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV-A59) contained autoantibodies (autoAb) that bound to a 40-kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross-reacted with a 40-kDa protein from human, rat and sheep liver, but not with liver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and the occurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV-infection. The 40-kDa protein was purified from mouse liver extracts by ion-exchange chromatography, gel filtration and SDS-PAGE. Because the N-terminal was blocked, we digested the protein in-gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.     

Determination and characterization of cross-reacting allergens in latex, avocado, banana, and kiwi fruit

Sera of 11 patients were used to characterize allergens in kiwi fruit, latex, avocado, and banana by SDS-PAGE/immunoblotting and to determine cross-reactions between these allergen extracts in EAST inhibition and immunoblot inhibition. By SDS-PAGE/immunoblotting. allergens with apparent molecular weights of 21, 38. 40. and 42 kDa were visualized in latex extract. In avocado extract. IgE-binding components of 27, 43, 52, 58, 65, 75, and 88 kDa were to be seen, whereas, in banana extract, a 40-kDa protein showed strong IgE binding. Furthermore, allergens of 52,58,88, and 94 kDa were detected in the extract of banana. Cross-reactions between these allergen extracts were determined by EAST inhibition. Immunoblot inhibition demonstrated that almost all IgE-reactive bands in nitrocellulose-blotted latex, avocado, and banana extracts and two components of 43 and 67 kDa in kiwi fruit shared common IgE epitopes.     

Effect of various stabilizing agents on Imperata cylindrica grass pollen allergen extract

BACKGROUND: Allergen extracts are unstable, heat labile or susceptible to proteases. Stability of allergen extracts is important for proper diagnosis and therapy of allergic disorders. OBJECTIVE: The present study was undertaken to determine the preservation and stabilization conditions of Imperata cylindrica (Ic) grass pollen extract. METHODS: The Ic extract was kept with 0.1 mepsilon-aminocaproic acid (EACA), 0.75 m sucrose, 5% glycerol, 0.03% human serum albumin (HSA) or 0.4% phenol for different time periods. The extracts were stored for 3, 6 and 12 months each at 4 degrees C, 4 degrees C with daily exposure to room temperature (RT) for 1 h, and RT. The quality of extracts was analysed by SDS-PAGE, Western blot, ELISA, ELISA inhibition and skin test. RESULTS: Extracts kept with EACA and sucrose retained most of the protein bands followed by glycerol as determined by SDS-PAGE and Western blot during all storage periods and conditions in comparison with standard extracts. The extracts kept with HSA, phenol and without preservative (WP) showed protein degradation below 33 kDa after 3 months storage at all conditions. However, a 67-kDa allergen was stable in these extracts. EACA extract required 75 to 120 ng of protein for 50% inhibition in IgE binding under different conditions, whereas standard extract required 70 ng for the same. ELISA also demonstrated high allergenic reactivity of EACA extract. ID test on allergy patients with EACA extract demonstrated same allergenic potency as that of standard extract. CONCLUSION: EACA is the best preservative/stabilizing agent of Ic pollen extract, followed by sucrose and glycerol. Ic extract kept with phenol, HSA and without preservative showed degradation within 3 months. EACA preserved extract is equally potent as that of standard extract up to 1 year's storage.     

Antineural antibodies in sera of leprosy patients.

A microtiter plate ELISA with semipurified human nerve sonicate antigen(s) (NA) was used to screen the sera of leprosy patients. High titers of IgG and low titers of IgM classes of antineural antibodies directed to peripheral nerve antigens were detected in LL, BL, BB, BT, and TT categories of leprosy. In the Western blot, leprosy sera recognized 50- to 55-, 85- and 108-kDa molecular weight protein bands of NA. The identity of these protein bands immunoreactive with leprosy sera was checked with a panel of commercially available antibodies to known neural proteins. The 50- to 55-kDa band reacted with anti-S100 and anti-glial fibrillary acidic protein antibodies while 85 and 108 kDa could not be identified. Whole immunoglobulins isolated from leprosy sera with high titers of antineural antibodies induced cytotoxicity of the cultured glial cell line in the presence of complement.     

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.     

Serodiagnosis of Lyme borreliosis by western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi.

The significance of various antibodies against Borrelia burgdorferi was studied by Western blot (immunoblot) by using 578 human serum samples. The proteins regularly detected by using samples from patients with Lyme borreliosis were those with bands with molecular masses of 94, 83, 75, 66, 60, 55, 46, 41, 39, 34, 31, 29, 22, and 17 kDa. The detectable frequencies of most of these proteins appeared to be significantly different between the sera from patients with Lyme borreliosis and those from normal control individuals as well as from the group with syphilis. The 39-kDa protein band recognized by polyvalent antibody was found to be the most significant marker for Lyme borreliosis. Furthermore, an anti-39-kDa immunoglobulin M response was detected in the samples from patients with early-stage Lyme borreliosis. Results from the use of monoclonal antibodies and patient sera revealed that the 39- and 41-kDa proteins may be structurally related but are immunologically distinct antigens. The significance of antibody reactivities to the 41-, 94-, 22-, 31-, and 34-kDa protein bands is also discussed.     

Enzyme immunoassay detection of antigen-specific immunoglobulin g antibodies in longitudinal serum samples from patients with cryptosporidiosis

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.     

Expression of gelatinise/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhancein vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinise/type IV collagenase in NE. Moreover, NE increased thein vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinise/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.     

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.     

Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease.

In October 1994, the Second National Conference on the Serologic Diagnosis of Lyme Disease recommended a two-step approach to serological testing. The first step was the performance of an enzyme-linked immunosorbent assay (ELISA); the second step was a confirmatory immunoblot. New criteria for the interpretation of a positive immunoblot were also recommended. The committee decided to omit the 31- and 34-kDa bands (OspA and OspB, respectively) from the choice of bands considered diagnostic for a positive immunoblot. Since we had previously included these in our diagnostic criteria for Lyme disease-positive immunoblots, we reviewed data for all patients attending a Lyme disease center with positive ELISAs and immunoblot assays for Lyme disease from 1 September 1992 to 31 December 1993. The criteria for a positive Western blot (immunoblot) were the presence of 5 or 12 bands, including the 10 recommended by the conference, and the presence of the 31- and 34-kDa protein bands. Of the 136 patients evaluated, 50 were considered to have Lyme disease. Of these 50, 4 (8%) would not have met immunoblot criteria for the diagnosis if the new recommendations were used. Had the 31- and 34-kDa bands been included as part of the diagnostic requirements for immunoblot, these patients would have been included. Although overdiagnosis of Lyme disease appears to be the more frequent problem, our concern is that the exclusion of the 31- and 34-kDa protein bands from the diagnostic criteria may result in the underdiagnosis of Lyme disease by those who would rely too heavily on serological confirmation. The addition of the 31- and 34-kDa bands to those recommended for confirmatory immunoblot should be reconsidered.     

Characterization of immunoglobulin G and its subclass response to Indian kala-azar infection before and after chemotherapy

Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogeneous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.     

Development and evaluation of a Western blot kit for diagnosis of human trichinellosis

We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.     

Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.     

Kinetics and diagnostic and prognostic potential of quantitative Western blot analysis and antigen-specific enzyme-linked immunosorbent assay in experimental canine leishmaniasis

Quantitative computerized Western blot analysis of antibody responses during experimental canine Leishmania infantum infection distinguished between immunodominant and nonimmunodominant protein bands. Six infected beagles, positive by both PCR and parasite culture, were monitored over 75 weeks postinfection and during a 12-week allopurinol treatment course. All dogs were symptomatic at the time of treatment. Of 12 antigenic bands examined, the immunodominant bands (12, 14, 24, 29, 48, and 68 kDa) showed significantly increased intensities (P<0.01) and higher frequencies of recognition than the nonimmunodominant bands at all time points. Detection of the former bands at 6 weeks postinfection preceded seroconversion by enzyme-linked immunosorbent assay (ELISA) both on crude Leishmania antigen or the recombinant proteins rK39 and HSP70. Reactivity with the 14-, 48-, and 68-kDa bands signified early infection, whereas increased reactivity with the 14-, 24-, and 29-kDa bands was associated with posttreatment parasite persistence and potential unfavorable prognosis. Total lane intensity (TLI) emerged as a sensitive marker for early infection and increased as early as 4 weeks postinfection. TLI had a significantly higher (P<0.01) relative increase rate than crude Leishmania antigen or HSP70 or rK39 ELISA at all time points. These immunodominant antigens and TLI, as determined by quantitative Western blotting, will be valuable for early detection and treatment evaluation of canine leishmaniasis.     

Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheria-tetanus-pertussis vaccine.

To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cell proteins of Bordetella pertussis after natural infection or vaccination with diphtheria-tetanus-pertussis vaccine. Serum specimens collected during a pertussis outbreak investigation and from uninfected persons were used in Western blot (immunoblot) analyses to determine the presence of immunoglobulin G (IgG) and IgA antibodies to specific B. pertussis proteins. IgG antibodies to proteins of molecular masses 220 and 210 kilodaltons (kDa) were detected in 14 of 18 serum samples obtained from patients with culture-confirmed pertussis greater than or equal to 40 days after the onset of coughing. IgA antibodies were detected in 15 of the 18 samples. Of 19 serum samples obtained from patients who had not been ill with pertussis, 6 contained IgG antibodies to these proteins and 1 contained IgA antibodies. The two proteins bound antiserum specific for filamentous hemagglutinin and comigrated with purified filamentous hemagglutinin. IgG antibodies to two additional protein bands of molecular masses 84 and 75 kDa were associated with previous vaccination. Antibody to the 84-kDa protein was detected in 15 of 17 vaccinated, never-infected persons, and antibody to the 75-kDa protein was detected in 16 of the 17. None of 11 nonvaccinated, never-infected persons tested had antibodies to either protein. All seven fully vaccinated persons with culture-documented infection had antibodies to both proteins. Antibodies to the 84-kDa protein were detected in 6 of 22 nonvaccinated and infected persons, and antibodies to the 75-kDa protein were detected in 8 of the 22. Use of Western blot analysis in this study allowed us to distinguish antibody responses to infection and immunization.