Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/10⁶ RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples.     

Serodiagnosis of human strongyloidiasis by an enzyme-linked immunosorbent assay

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of strongyloidiasis has been investigated. 45 men with long-standing strongyloidiasis were compared with the same number of age-and sex-matched control subjects. The ELISA detected antibody in 84% of patients with parasitologically proven strongyloidiasis. When the technique was compared with an indirect immunofluorescent assay (IFA), a high correlation coefficient was obtained. Specificity was demonstrated by observing a marked fall in optical density of pooled positive serum after prior incubation with Strongyloides ratti soluble antigen but not after incubation with antigens derived from Ascaris suum or Dirofilaria immitis. The test is simple and offers a useful method for the diagnosis of strongyloidiasis. In these patients it was more reliable than a single parasitological examination of faeces or duodenal contents.     

A multi-species malaria antigen for use in the indirect fluorescent antibody test

A multi-species thick smear antigen containing equal proportions of P. vivax, P. falciparum, and P. brasilianum schizonts was prepared for use in the indirect fluorescent antibody test for malaria. Tests with 80 sera showed that antibody titres with the multi-species antigen always equalled or exceeded the highest titre obtained when the sera were tested with the constituent mono-species antigens. Use of the multi-species antigen greatly reduces the time and labour required to screen serum specimens for malaria antibody. This study confirms the need for homologous antigens of all species to be detected if maximum sensitivity and reactivity are to be achieved.     

Diagnostic Accuracy of the E-Plate Serum Antibody Test Kit in Detecting Helicobacter pylori Infection Among Japanese Children

Background

A number of noninvasive diagnostic tests are available to detect Helicobacter pylori infection. Data on serologic testing of children are lacking, however, and thus it remains unclear whether the serology cutoff points used for adults are appropriate for children.

Methods

Serum and stool samples were obtained from 73 children who visited 5 hospitals in Japan between March 1993 and December 2009. Analysis of stool samples was carried out using an H pylori stool antigen enzyme-linked immunosorbent assay (HpSA ELISA), and serum antibodies to H pylori were examined using an antibody determination kit (E-Plate Eiken H pylori antibody). The validity of the serologic test was evaluated based on its sensitivity, specificity, and receiver operating characteristics curve.

Results

Of the 73 children included in this study, 34 were HpSA-positive and 39 were negative. Among the 34 HpSA-positive patients, 32 were IgG-positive and 2 were IgG-negative. Of the 39 patients who were HpSA-negative, 38 were IgG-negative and 1 was IgG-positive. The sensitivity, specificity, and positive likelihood ratio for IgG antibody testing were 91.2%, 97.4%, and 35.6, respectively, based on the recommended adult cutoff point of 10 U/ml. Among children, use of cutoff points in the range of 7 to 9 U/ml yielded optimal values for sensitivity and specificity, as well as a positive likelihood ratio.

Conclusions

The performance of the E-plate anti-H pylori IgG antibody test was comparable to that of the stool antigen test and is therefore suitable for epidemiologic studies of H pylori infection in large samples.Key words: Helicobacter pylori, serologic test, validity, stool antigen test     

Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay for the detection of malaria antigens

A serodiagnostic test has been developed for the detection of Plasmodium falciparum in infected blood. Using parasite antigens and infected red blood cells from in vitro cultures of P. falciparum and malaria antibody from high-titre Gambian sera, parasites were detected in a solid-phase radioimmunoassay (RIA) that measured antibody-binding inhibition. Lysed RBC were incubated with labelled IgG purified from immune sera and were then placed in antigen-coated microtubes and incubated. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test RBC and, using dilutions of RBC from in vitro cultures of P. falciparum, the test detected infection at a level of 8 parasites/10⁶ red blood cells. The test was applied to RBC from 100 healthy European blood donors and to samples of RBC from 500 Gambians from the up-country villages of Keneba and Manduar. More samples were positive by RIA than by microscopy and there was a highly significant degree of correlation between the RIA and microscopy results.     

The Use of Recombinant 31 kDa Antigens of Trichinella spiralis for Serodiagnosis of Experimental Trichinellosis

Background:

We have previously reported that a 31 kDa protein was screened from the excretory-secretory (ES) proteins of Tichinella spiralis muscle larvae (ML) by immunoproteomics using early infection sera, and the gene encoding a 31 kDa protein from T. spiralis was cloned and expressed in an E. coli expression system. In this study, the recombinant 31 kDa antigens were used for detection of anti-Trichinella antibodies in serum of experimentally infected mice by ELISA.

Methods:

Anti-Trichinella IgG antibodies in sera of mice infected with Trichinella were assayed by ELISA with recombinant 31 kDa antigens, and its sensitivity and specificity were compared with ELISA with ES antigen.

Results:

The sensitivity and specificity of ELISA with recombinant antigens was 96.67% (29/30) and 96.87% (62/64), compared with 100% (30/30) and 98.44% (63/64) of ELISA with ES antigens was (P>0.05). In heavily, moderately and lightly infected mice (500, 300 and 100 larvae/mouse), anti-Trichinella antibodies were firstly detected by ELISA with recombinant antigens at 8, 12 and 14 dpi, respectively; then increased rapidly with a detection rate of 100% respectively at 28, 22 and 30 dpi. While the antibodies were firstly detected by ELISA with ES antigens at 10, 8 and 10 dpi, respectively, the antibody positive rate reached 100% at 14, 12 and 22 dpi, respectively.

Conclusion:

The recombinant 31 kDa antigens of T. spirali had a good sensitivity and specificity for detecting anti-Trichinella antibodies and might be the potential diagnostic antigen for trichinellosis.     

幽门螺杆菌全菌抗原与尿素酶纯化抗原ELISA检测在人群血清学诊断中的应用

用纯化的Ｈｐ抗原对６７６例各型胃病患者进行了血清抗幽门螺杆菌尿素酶抗体的检测。实验证明其特异性为９６％，敏感性为９８％。血清学ＥＬＩＳＡ检测法，尤其适宜于流行病学调查，还可用于药物治疗效果的观察和筛选非溃疡性消化不良。     

Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity

Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response.     

Variability of in vivo potency assays of whole-cell pertussis,inactivated polio,and meningococcal B vaccines

For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing.     

Research on ascariasis immunity and immunodiagnosis

The pattern of antibody response to Ascaris in the blood of experimentally infected human beings, pigs and laboratory animals indicates that immunity to ascariasis develops mainly under the effect of migrating larvae. The antibody can be demonstrated in the microprecipitation test on live larvae in vitro and in the agglutination test with carmine-adsorbed antigen. The antibodies can be detected 5-10 days after infection and remain in the blood for 90-100 days.     

An evaluation of the ELISA for schistosomiasis in a hospital population

In order to evaluate the ELISA for schistosomiasis under the conditions of clinical practice, 1576 hospital patients were tested using a crude soluble Schistosoma mansoni egg antigen. Test sensitivity in detection of S. mansoni was found to be 96·2% and in S. haematobium 92·3%. The predictive value of positive results was high, reaching 88% at antibody levels three or more times the screening level. The test was considered by clinicians to be valuable for diagnosis and patient management, though it did not distinguish active from recently treated infections. Of 37 apparently false positive schistosome ELISA results only seven could be attributed to other helminth infections. Another nine patients had hepatitis. It is suggested that the antigens and antibodies of the two diseases are mutually cross-reactive, since reports have suggested a high increase of HBsAg patients with schistosomiasis.     

Immunodiagnosis of parasitic zoonoses: sensitivity and specificity of in vitro lymphocyte proliferative responsiveness using nematode antigens purified by affinity chromatography

The sensitivity of in vitro lymphocyte proliferative responsiveness using antigens purified by affinity chromatography, was greater than that of three serological tests and allowed the specific identification of Dirofilaria immitis, Toxocara canis, Angiostrongylus cantonensis and Ascaris lumbricoides in animals sensitized with parasite antigens, and the diagnosis of natural (D. immitis, T. canis and Angiostrongylus cantonensis) and zoonotic nematode infections.Lymphocytes remained viable in whole blood suspensions in RPM 1640 in a polystyrene insulation box at air temperature for at least 12 hours and reacted in the cell-mediated immunity test (CMIT) without loss of response.The CMIT proved useful in the early immunodiagnosis of nematode infections and showed that parasite antigens purified by affinity chromatography retained a high degree of specificity and sensitivity in both the CMIT and serological tests.     

Detection of specific antibodies to pigeon serum and bloom antigens by enzyme linked immunosorbent assay in pigeon breeder's disease

BACKGROUND—Pigeon breeder's disease is an extrinsic allergic alveolitis in the lungs of sensitised people, caused by hypersensitivity reactions to inhaled pigeon antigens. Antigens from different sources of the animal are used for diagnostic purposes, with serum being the most widely used. Bloom is rarely used; very little is known of its antigenicity and diagnostic performance, particularly when used with the enzyme linked immunosorbent assay (ELISA) method, which is the most popular test as it permits measurement of the antibody response.
METHODS—To (a) standardise an ELISA for the measurement of specific IgG against pigeon serum and pigeon bloom extract; (b) to establish reference values for specific IgG in 73 non-exposed controls, (c) to show the presence of specific IgG against pigeon serum and bloom in serum samples of 17 patients with bird fancier's lung and 11 asymptomatic fanciers, and (d) to study the similarity of the two antigen sources by cross reactivity experiments.
RESULTS—Reference values of specific IgG were defined with the 97.5 percentile (367.9 U/ml for pigeon serum and 953.7 U/ml for pigeon bloom extract). Of symptomatic patients 100% had values higher than the cut off for both antigens. In asymptomatic fanciers values were higher than the cut off for pigeon serum in 45% and bloom extract in 54%. Cross reactivity experiments showed that the two antigens differed in antigenic content although some components may be common to both.
CONCLUSION—The ELISA methods used proved to be useful tools for evaluating specific IgG antibody responses against both antigens. The diagnostic performance of both ELISA methods performed with these antigen sources was similar, showing very high sensitivity but moderate specificity. Although some antigenic similarity was found between pigeon serum and bloom extract, cross reactivity studies showed that various antigens seemed to be specific to the bloom extract. However, the antigens responsible for pigeon breeder's disease seem to be present in both antigenic sources.


Keywords: antibodies; pigeons; ELISA     

Precipitating antibody response to malarial S-antigens

The gel diffusion test has been used to detect antibodies to malarial S-antigens. Sera were obtained from entire Gambian village communities, from young children with acute P. falciparum malaria, from children convalescent from such infections and from immune adults. In community studies, small selections of S-antigens detected antibody frequently in sera from older persons but rarely in sera from young children. Larger panels of antigens detected antibodies in sera from half of 50 malarious children while homologous antibody responses were observed in 22% of 267 children followed at intervals during convalescence from malaria. In these latter cases, antibody tended to appear more swiftly when antigen was lost rapidly from the circulation, and observations made on individual responses indicated that antibody production was influenced by factors other than the intrinsic properties of the antigens. In adult sera antibodies usually occurred in association with IgG.     

Immunogenicity of a recombinant fusion construct composed of intrinsically unstructured,low polymorphic segments derived from merozoite surface protein 2 and trophozoite exported protein 1

To overcome the extensive polymorphism found in human Plasmodium antigens and to avoid the lengthy characterization of their 3 dimensional structure and subsequent production of the native proteins we have been concentrated in large unstructured, non-or low-polymorphic fragments present in the blood stage of P. falciparum. Three fragments derived from the 2 family-specific and constant regions of merozoite surface protein (MSP2) and PFF0165c protein were previously selected for evaluation as potential single vaccine candidates. In order to increase and optimize their potential efficacy against P. falciparum infection the 3 antigens were combined in a single DNA recombinant product (FusN) and compared its antigenicity with that of single antigens in sera of volunteers living in endemic countries. Immunogenicity of the FusN was then compared with that of the mixture of 3 antigens in 3 strains of mice. Antigen specific, affinity purified human antibodies were then tested in antibody dependent cellular inhibition and merozoite opsonization assays. In addition, the antigen specific antibody response and its association with protection from malaria infection were determined. The data collected indicate that the recombinant product is an equal or better antigen /immunogen than fragments used either alone or as a mixture for vaccination in combination with adjuvant. In addition, antibody response to FusN shows a stronger association with protection than single fragments. The use of a single construct as vaccine would drastically reduce the cost of manufacturing and development of the GMP product.     

Immunogenicity of the Lyme disease antigen OspA,particleized by cobalt porphyrin-phospholipid liposomes

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.     

Further characterization of filarial antigens by SDS polyacrylamide gel electrophoresis

SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis of an antigen isolated from sera of Wuchereria bancrofti-infected patients and Setaria digitata antigen SD2-4 is reported. Both antigens showed carbohydrate (glycoprotein) staining. The W. bancrofti antigen had an apparent relative molecular mass of 35 000 while the S. digitata antigen SD2-4 migrated at the marker dye position on SDS-polyacrylamide gel electrophoresis. SDS treatment of these antigens did not abolish the precipitation reaction with antibody. In the case of W. bancrofti antigen, SDS treatment probably exposed hitherto hidden antigen epitopes.     

The cAMP assay: A functional in vitro alternative to the in vivo Histamine Sensitization test

Safety requirements stipulate the performance of the in vivo Histamine Sensitization (HS) test for quality control of acellular pertussis (aP) vaccines. For reasons of reproducibility and animal welfare concern, an in vitro assay was developed. The assay reflects the mechanism of histamine sensitization and is based on cAMP production in A10 cells to residual pertussis toxin (PT). We showed that PT induces cAMP levels in a dose-dependent manner while the sensitivity of the assay equals the sensitivity of the HS test. Neither the individual components nor the combination vaccine DTaP-IP did affect the assay. The cAMP assay meets the criteria for specificity and sensitivity and therefore might be a promising candidate to replace the HS test.     

Induction of TLR4-dependent CD8+ T cell immunity by murine β-defensin2 fusion protein vaccines

Our laboratory previously described the strategy of fusing chemokine receptor ligands to antigens in order to generate immunogenic DNA vaccines. In the present study, we produced mouse β-2 defensin (mBD2) fusion proteins using both ovalbumin (OVA) and gp100 as model antigens. Superior cross-presentation by dendritic cells (DC) was observed for mBD2 fused antigens over unfused antigens in vitro. In vivo, we observed significant increases in the expansion of adoptively transferred antigen-specific MHC class I, but not class II-restricted T cells after immunization with mBD2 fused antigen over antigen alone. This enhanced expansion of class I restricted T cells was Toll-like receptor 4 (TLR4) dependent, but CC chemokine receptor 6 (CCR6) independent. Superior tumor resistance was observed for mBD2-fusion protein vaccines, compared to unfused antigen, in both B16-OVA and B16 tumor models. These data suggest that production of mBD2 fusion proteins is feasible and that the vaccines facilitate in vivo expansion of adoptively transferred T cells through a TLR4-dependent mechanism.