Antibody Capture Enzyme-linked Immunosorbent Assay Research Articles

Dose–response and time course relationships for vitellogenin induction in male western fence lizards (Sceloporus occidentalis) exposed to ethinylestradiol

The long-term goal of this research is to develop and validate an in vivo reptile model for endocrine-mediated toxicity using fence lizards (Sceloporus spp.). One of the best defined estrogenic responses in oviparous vertebrates is induction of the yolk precursor protein, vitellogenin (Vtg). In this study, dose-response and time course relationships for Vtg induction were determined in male western fence lizards (Sceloporus occidentalis) given intraperitoneal injections of 17alpha-ethinylestradiol (EE2). Plasma Vtg was quantified directly with an antibody-capture enzyme-linked immunosorbent assay (ELISA) and indirectly using plasma alkaline-labile phosphate (ALP) in order to compare these two methods. Both ELISA and ALP predicted similar median effective dose (ED50 [dose causing a 50% maximal response]) values for plasma Vtg induction (0.167 mg/kg for ELISA and 0.095 mg/kg for ALP). In addition, both ELISA and ALP detected significant Vtg induction at a dose of 0.0003 mg/kg of EE2, which was the lowest dose used in our study. A decrease in body weight at the highest dose (10 mg/kg) and an increase in hepatosomatic index at the four highest doses were observed. Serial dilutions of plasma from an EE2-exposed male revealed a high correlation between plasma Vtg and ALP determinations in this species. In conclusion, our data show that plasma ALP may be a suitable alternative for measuring plasma Vtg compared with developing a Vtg ELISA in fence lizards exposed to estrogenic compounds.

Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States.

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.

Bone marrow samples from patients with aplastic anemia are not infected with parvovirus B19 and Mycobacterium tuberculosis.

The majority of patients with aplastic anemia (AA) have an idiopathic form of the disease. The aim of this study was to detect the presence of parvovirus B19 DNA and Mycobacterium tuberculosis (MTB) DNA by nested polymerase chain reaction (N-PCR) assays in the bone marrow biopsy samples from 30 patients with idiopathic AA. Serologic assays for parvovirus B19 were based on indirect antibody capture enzyme-linked immunosorbent assay. Our results indicate that neither parvovirus B19 DNA nor MTB DNA could be demonstrated in any of the bone marrow samples by N-PCR. Moreover, IgM antibody against parvovirus B19 also was undetectable in the serum samples of 17 patients. Thus, our results suggest that parvovirus B19 and MTB are not associated with AA and, consequently, do not have a role in the pathogenesis of this disease.

A recombinant particulate antigen of Japanese encephalitis virus produced in stably-transformed cells is an effective noninfectious antigen and subunit immunogen

A COS-1 cell line, stably transformed by a plasmid encoding the premembrane and envelope glycoproteins of Japanese encephalitis virus, produced a noninfectious recombinant antigen expressed as extracellular particles. Extracellular particles purified by equilibrium density centrifugation in sucrose gradients followed by electron microscopy were characterized as spherical particles with an average diameter of approximately 30 nm and a buoyant density of 1.15 g/cc. Purified extracellular particles were shown by western blot to contain premembrane, membrane and envelope proteins. The gradient-purified particles exhibited hemagglutination activity at the same pH optimum (6.6) as Japanese encephalitis virus. Recombinant antigen from cell culture fluid was concentrated by precipitation with polyethylene glycol and evaluated for immunogenicity in 8–10-week-old ICR mice. Groups of five mice received only one immunization of recombinant antigen with or without Freund's incomplete adjuvant. Mice immunized with recombinant antigen plus Freund's incomplete adjuvant elicited the highest anti-viral titers as determined by both enzyme-linked immunosorbent assay (ELISA) and plaque-reduction neutralization tests. The polyethylene glycol-concentrated recombinant antigen was also evaluated for use in IgM antibody-capture ELISA and indirect IgG ELISA. The IgM-capture ELISA results using recombinant antigen correlated well with the results of a similar test using Japanese encephalitis virus-infected mouse brain antigen for the analysis of serum samples from patients with symptoms of acute encephalitis. Similar IgG titers were observed in an indirect ELISA comparing recombinant antigen and purified Japanese encephalitis virus as plate-bound antigens. Based on these studies, this entirely safe, easily produced antigen that expresses authentic Japanese encephalitis virus envelope glycoprotein would provide an excellent alternative to standard viral antigens used in various ELISA formats.

Fully Automated Anti-Varicella Zoster Enzyme-Linked Immunosorbent Assays Supporting Manufacture of a Novel Intravenous Therapeutic for Treatment of Ailments Caused by the Varicella Zoster Virus

Two non-competitive, antibody-capture enzyme-linked immunosorbent assays, a commercial Kit VZIG ELISA and an in-house developed, amplified VZIG ELISA were automated by using the liquid handling and plate processing abilities of the Microlab AT plus 2 and Microlab F.A.M.E. instruments, respectively. The evaluation of the experimental data has also been automated by using the F.A.M.E.-specific data import converter and PLA software program for data capture / uploading and analysis. The automation increased the economy of analytical operations and traceability of the experimental data thus better supporting the production and clinical studies on a therapeutic Varicella Zoster Immune Globulin manufactured at Cangene.

West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.

Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

Fully Automated Anti-Varicella Zoster Enzyme-Linked Immunosorbent Assays Supporting Manufacture of a Novel Intravenous Therapeutic for Treatment of Ailments Caused by the Varicella Zoster Virus

Two non-competitive, antibody-capture enzyme-linked immunosorbent assays, a commercial Kit VZIG ELISA and an in-house developed, amplified VZIG ELISA were automated by using the liquid handling and plate processing abilities of the Microlab AT plus 2 and Microlab F.A.M.E. instruments, respectively. The evaluation of the experimental data has also been automated by using the F.A.M.E.-specific data import converter and PLA software program for data capture / uploading and analysis. The automation increased the economy of analytical operations and traceability of the experimental data thus better supporting the production and clinical studies on a therapeutic Varicella Zoster Immune Globulin manufactured at Cangene.

Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay.

We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.

Detection of Antibody to Turkey Coronavirus by Antibody-Capture Enzyme-Linked Immunosorbent Assay Utilizing Infectious Bronchitis Virus Antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.

HIV-1 prevalence, HIV-1 subtypes and risk factors among fishermen in the Gulf of Thailand and the Andaman Sea.

Mobile populations are thought to be at high risk for HIV-1 infection. This study aims to determine the prevalence of HIV-1 infection, HIV-1 subtypes and socio-demographic and behavioural risk factors among fishermen in the Gulf of Thailand and the Andaman Sea. A cross-sectional survey, consisting of face-to-face interviews and the collection of oral fluid samples, was conducted in Samut Sakorn, Ranong, Songkhla and Traat Provinces, Thailand, between January and April 1998. Oral fluid samples were double-tested for HIV-1 antibody by IgG antibody capture enzyme-linked immunosorbent assay and enzyme immunoassay, and confirmed by Western blot. The presence of subtypes B' and E was assessed using a peptide enzyme immunoassay. Of the 818 fishermen (582 Thai, 137 Burmese, 99 Khmer) 15.5% were HIV-1 positive: 14.6% among Thai, 16.1% among Burmese and 20.2% among Khmer. Of the 119 HIV-1 positive samples available for subtyping, 72 (61%) were subtype E, 15 (13%) were subtype B'; the subtype could not be determined for 32 (27%) samples. Sixteen per cent of subjects had ever visited a commercial sex worker outside Thailand. This behaviour was more prevalent among Khmer (40%) than among Thai and Burmese (12%). In univariate logistic regression analysis, being 25 to 32 years of age, compared with being older or younger; working as a fisherman between 4 and 10 years, compared with working for a shorter or longer period; being unmarried; being a steersman or mechanic, compared with being a skipper or ship hand; greater number of visits to commercial sex workers; having visited a commercial sex worker outside Thailand; alcohol or drug use before or during sex; being tattooed; and having a history of sexually transmitted disease were significantly related to prevalent HIV-1 infection. Male-to-male sex and injection drug use were rarely reported in this population. In multivariate analysis, being 25 to 32 years of age, being unmarried, having a tattoo and a greater number of visits to commercial sex workers remained in the model to predict HIV-1 prevalence. A history of drug injection was predictive for infection with HIV-1 subtype B'. These findings indicate a high HIV-1 prevalence among fishermen in the Gulf of Thailand and the Andaman Sea. Risk factor analysis suggests that heterosexual intercourse is the major mode of transmission in this population. Increased efforts to reduce the spread of HIV-1 among this epidemiologically important group are urgently needed.

Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections.

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of >/=2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

Serological diagnosis of varicella–zoster virus in sera with antibody-capture enzyme-linked immunosorbent assay of IgM

Evaluation was made of three enzyme-linked immunosorbent assay (ELISA) formats; varicella–zoster virus (VZV) indirect ELISA; VZV IgM capture using biotin and VZV IgM capture using peroxidase, for the detection of VZV-specific IgM antibodies in human sera. It was observed that there was no significant difference in sensitivity of detection using the three formats but there were important practical differences in the number of steps and hence time for assay completion between the three assay formats. All assays showed some cross-reactivity with sera containing anti-HSV1 antibodies.

Apolipoprotein A-I in bile inhibits cholesterol crystallization and modifies transcellular lipid transfer through cultured human gall-bladder epithelial cells.

Apolipoprotein A-I (Apo A-I), conventionally purified by several steps including organic solvent-delipidation from plasma, inhibits cholesterol crystallization in bile. To observe a significant effect in vitro, however, supraphysiological concentrations above 100 microg/mL are required. For this reason, this protein has not been considered to play a physiological role in vivo. In the present study, we examined the cholesterol crystal growth-inhibiting effect of biliary Apo A-I at its physiological concentration, the modification of transcellular transfer of biliary lipids through cultured human gall-bladder epithelial cells (GBEC) by Apo A-I at its physiological concentration and the binding and secretion of Apo A-I by GBEC. We purified biliary Apo A-I to near homogeneity using immobilized artificial membrane chromatography. At 5 microg/mL, biliary Apo A-I reduced cholesterol crystal mass by 50%, whereas plasma-derived, solvent-delipidated Apo A-I had no effect. Using an antibody-capture enzyme-linked immunosorbent assay, we found reduced Apo A-I concentrations in bile samples from gallstone patients when compared with bile samples from gallstone-free controls (medians, 2.35 and 9.4 microg/mL, respectively). In a GBEC line, Apo A-I (5 microg/mL) enhanced transfer of phospholipid and cholesterol from the mucosal to the serosal side of cell monolayers by approximately 50%. These cells appear to bind Apo A-I reversibly in a dose- and time-dependent manner, compatible with receptor-type binding. Cultured human gall-bladder epithelial cells also showed basal secretion of Apo A-I, which was greatly increased by exposure to model bile solutions. Apolipoprotein A-I in bile, thus, has both a direct effect on cholesterol crystal formation and enhances lipid removal from gall-bladder bile by GBEC. This effect may be specific and receptor mediated. These observations support two separate roles for human biliary Apo A-I and suggest that this protein may be important in preventing the formation of cholesterol crystals (the initial step in gallstone formation) in supersaturated bile.

Seroepidemiology of human parvovirus B19 in Taiwan

In order to determine the prevalence and risk factors of human parvovirus B19 (B19) infection in Taiwan, a seroepidemiological study was carried out in 19 townships. Serum samples were collected from 862 healthy residents, who were selected by stratified random sampling from various study areas. They were chosen from four different ethnic groups including aborigines, Fukien Taiwanese, Hakka Taiwanese, and mainland Chinese. Serum samples were screened for B19 IgG antibody by indirect antibody capture enzyme-linked immunosorbent assay (ELISA) and B19 IgM by IgM antibody capture (MAC)-ELISA, respectively. The overall prevalence of anti-B19 IgG and anti-B19 IgM was 32.8% and 0.35%, respectively. The anti-B19 seropositive rate in females was significantly higher than that of males (36.4% vs. 29.4%, P < .001). The age-sex-adjusted seropositive rate in urban townships (39.9%) was higher than that in aboriginal townships (30.5%, P < .001). The seropositive rate increased significantly with age showing a dose-response relationship (P=0.0001 based on a trend test). Blood transfusion was found to be associated with an increased seropositive rate showing a multivariate-adjusted odds ratios of 1.6.

Seroepidemiology of human parvovirus B19 in Taiwan

In order to determine the prevalence and risk factors of human parvovirus B19 (B19) infection in Taiwan, a seroepidemiological study was carried out in 19 townships. Serum samples were collected from 862 healthy residents, who were selected by stratified random sampling from various study areas. They were chosen from four different ethnic groups including aborigines, Fukien Taiwanese, Hakka Taiwanese, and mainland Chinese. Serum samples were screened for B19 IgG antibody by indirect antibody capture enzyme-linked immunosorbent assay (ELISA) and B19 IgM by IgM antibody capture (MAC)-ELISA, respectively. The overall prevalence of anti-B19 IgG and anti-B19 IgM was 32.8% and 0.35%, respectively. The anti-B19 seropositive rate in females was significantly higher than that of males (36.4% vs. 29.4%, P < .001). The age-sex-adjusted seropositive rate in urban townships (39.9%) was higher than that in aboriginal townships (30.5%, P < .001). The seropositive rate increased significantly with age showing a dose–response relationship (P = 0.0001 based on a trend test). Blood transfusion was found to be associated with an increased seropositive rate showing a multivariate-adjusted odds ratios of 1.6. J. Med. Virol. 57:169–173, 1999. © 1999 Wiley-Liss, Inc.

Gag (Mycteroperca microlepis) vitellogenin: purification, characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper

Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), can be used as a biochemical indicator of maturation in female fish. Here we report on purification and partial characterization of VTG from a temperate marine serranid, the gag(Mycteroperca microlepis). Development of a competitive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is also described. The gVTG was purified by DEAE-agarose anion exchange chromatography from a pooled plasma sample collected from several juvenile gag after they were injected with 17βestradiol. The protein appeared as a major band of Mr≅183 000 after SDS-PAGE ± Western blotting using either a specific rabbit antiserum to gVTG or a universal monoclonal antibody for vertebrate VTGs. Amino acid composition analysis and N-terminal peptide sequencing verified that gVTG is similar in primary structure to VTG from several other teleost species. The purified gVTG and its specific antiserum were used to develop a sensitive, competitive, antibody-capture ELISA for quantifying the protein in blood plasma from maturing females. VTG levels in maturing female gag were highly correlated with oocyte growth and circulating testosterone and 17β-estradiol levels, whereas VTG was non-detectable in juveniles, immature females or males. Two size-based maturity schedules for female gag were constructed, one utilizing detection of VTG in their circulation as a marker of maturity and the other relying on histological evidence that their ovaries were in vitellogenic or later stages of maturation. The two schedules were virtually identical. The gVTG ELISA was also used to detect VTG in blood plasma from mature Nassau grouper (Epinephelus striatus) and red hind (E. guttatus). As with gag, the assay was completely reliable for discriminating between reproductively mature females versus males from these two grouper species.

Analysis of immunoglobulin A antibodies to Helicobacter pylori in serum and gastric juice in relation to mucosal inflammation.

Helicobacter pylori is a major etiologic agent in gastroduodenal disorders. In this study, immunoglobulin A (IgA) antibodies to H. pylori antigens were evaluated in serum and gastric juice specimens obtained from patients with gastritis or peptic ulcers by utilizing antibody capture enzyme-linked immunosorbent assays (ACELISAs). Urease alpha subunit (UA), urease beta subunit (UB), the 66-kDa heat shock protein (HSP), and the 25-kDa protein (25K) were used as antigens for the ACELISAs. The antibody titers of the ACELISAs reflect the ratio of H. pylori-specific IgA to total IgA. The ratio is stable, although the antibody concentration fluctuates in gastric juice. By using ACELISAs it was possible to evaluate quantitatively not only serum IgA antibodies but also gastric juice secretory IgA (S-IgA) antibodies. In both serum IgA and gastric juice S-IgA ACELISAs, the titers of antibody to HSP and 25K were remarkably correlated with the histologic grade of gastritis, whereas those to UA and UB were not strongly correlated with histologic grade. Thus, it is useful for estimating the histologic grade of gastritis to quantify serum IgA and gastric juice S-IgA antibodies to HSP and 25K.

Further validation of antibody-capture and antigen-capture enzyme-linked immunosorbent assays for determining exposure of cattle to bovine coronavirus

Abstract Objective To further validate an antibody-capture ELISA for measuring bovine coronavirus (BCV) exposure (antibody seroresponse) in cattle and to explain the apparent loss of sensitivity of a BCV antigen-capture ELISA when testing feces from adult versus neonatal cattle. Animals 98 adult cows from herds with and without winter dysentery; 10 gnotobiotic or colostrum-deprived calves. Procedures Results of an antibody-capture ELISA for BCV and a plaque reduction virus neutralization assay performed on paired serum samples from 24 cattle were compared with each other and with results of immunoelectron microscopy (IEM) of feces for BCV. For samples from 98 cattle, results of antibody-capture ELISA were compared with results of IEM. Calves were inoculated with feces ELISA-positive or IEM-positive for BCV and monitored for BCV infection. An ELISA was developed to detect BCV antigen-antibody complexes in feces and results were compared with results of an antigen-capture ELISA and IEM. Results Antibody-capture ELISA results correlated with neutralization assay results, but agreed more closely with results of IEM. Calves became infected with BCV following inoculation with either ELISA-positive or ELISA-negative but IEM-positive feces. Results of the antigen-antibody ELISA correlated with results of IEM and the antigen-capture ELISA. Clinical Implications In adult cattle, testing of paired serum samples by use of an antibody-capture ELISA may be a better indicator of recent BCV exposure than results of virus neutralization tests. Antigen-antibody binding in feces may interfere with results of antigen-capture ELISA for BCV. (Am J Vet Res 1998;59:956–960)

Quantitation of cat immunoglobulins in the hemolymph of cat fleas (Siphonaptera: Pulicidae) after feeding on blood.

Passage of ingested cat immunoglobulin G (IgG) into the hemocoel of cat fleas, Ctenocephalides felis (Bouché), was examined using antibody capture enzyme-linked immunosorbent assays (ELISA) and Western blotting. Fleas were fed heparinized cat blood via membrane feeders. Cat IgG was present in the hemolymph of engorged female fleas 1 h after ingestion at an estimated quantity of 35 +/- 14 micrograms/ml. The prevalence of fleas with demonstrable cat IgG in their hemolymph 1 h after feeding was 100% for both female and male fleas. Following a single blood meal, cat IgG was present in the hemolymph of all 15 fleas tested 1 h after ingestion but dissipated below detectable levels in 10 of 20 fleas examined 3 h after ingestion, and was detectable in only 1 of 10 fleas examined 18 h after ingestion. However, when fleas were provided with continual access to blood over a 72-h period, IgG content in hemolymph, as measured in excised, triturated legs of individual fleas, remained fairly constant (3-16 pg IgG per sample). Flea feeding studies using specific antisera indicated that IgG in flea hemolymph retained its binding activity, and that at least a portion of the IgG was intact. Passage of ingested host antibody from gut into hemocoel is a prerequisite for the possible development of antiflea vaccines that target antigens outside of the flea midgut lumen (e.g., key components of the flea endocrine system controlling oogenesis).

IgM and IgA antibody responses in 12 cases of human acquired toxoplasmosis.

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels < or = 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis.