应用12D5和2187单克隆抗体检测广州管圆线虫循环抗原的研究

目的研制快速诊断广州管圆线虫感染的金标层析检测试剂。方法双抗体(12D5和2187)夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管网线虫感染病例血清循环抗原(CAg),同时将12D5和2187单抗点样于同相的硝酸纤维膜上,以胶体金-proteinA为标记物制备金标免疫层析试剂快速检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病例血清循环抗原。结果经鉴定单抗12D5为IgGl,2187为IgM,两株单抗同时识别广州管围线虫成虫相对分子质量55×103的蛋白,12D5和2187双抗体夹心ELISA和金标层析法对实验感染广州管网线虫的大鼠血清中CAg检出率为100％(48/48),广州管囤线虫感染病例血清CAg检出率为100％(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病例血清无交叉反应,与健康人血清无反应。结论12D5、2187双抗体夹心ELISA和金标层析法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,金标层析法操作简便、快速,结果判读容易,不需特殊设备,且敏感性高,能够确定现症感染。     

应用12D5和21B7单克隆抗体检测广州管圆线虫循环抗原的研究

目的 研制快速诊断广州管圆线虫感染的金标层析检测试剂.方法 双抗体(12D5和21B7)夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病例血清循环抗原(CAg),同时将12D5和21B7单抗点样于固相的硝酸纤维膜上,以胶体金-protein A为标记物制备金标免疫层析试剂快速检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病例血清循环抗原.结果经鉴定单抗12D5为IgG1,21B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量55×10~3的蛋白,12D5和21B7双抗体夹心ELISA和金标层析法对实验感染广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病例血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病例血清无交叉反应,与健康人血清无反应.结论 12D5、21B7双抗体夹心ELISA和金标层析法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,金标层析法操作简便、快速,结果判读容易,不需特殊设备,且敏感性高,能够确定现症感染.     

Detection of serum antibodies in human Hymenolepis infection by enzyme immunoassay.

Although hymenolepiasis is the commonest cestode infection of man, there are no data available on the human immune response to this parasite. Thus, in order to determine if infection induces antibodies against Hymenolepis nana antigens, sera from 52 infected children were initially studied on Ouchterlony plates and then by enzyme-linked immunosorbent assay (ELISA), using a crude antigenic extract prepared from scolex and neck regions of adult worms. In addition, sera from persons with cysticercosis, taeniasis and other parasitoses, and normal human sera, were studied. Only one serum from the Hymenolepis group showed precipitin antibodies against H. nana antigen, while several were positive by ELISA. The sensitivity of the ELISA was 84.62% and its specificity was 100%. Very high cross-reactivity rates were obtained with taeniasis (70.6%) and cysticercosis (75%) sera. These results show that Hymenolepis infection in man induces a low but detectable humoral immune response. Although not useful for diagnostic purposes, this may be relevant to the serodiagnosis of other tissue cestode infections of man, since antibodies detected in serological tests used for the diagnosis of cysticercosis, and probably hydatidosis, could be induced by H. nana instead of Taenia solium or Echinococcus larvae.     

Detection of chicken antibodies to mosquito salivary gland antigens by enzyme immunoassay

Sentinel chickens are used to detect western equine encephalomyelitis, St. Louis encephalitis, and West Nile virus activity. Flocks that receive high mosquito exposure will be most effective for surveillance purposes. However, mosquito population indices at the flock sites may only provide an indirect measure of potential exposure. Therefore, we developed an indirect enzyme immunoassay to detect chicken antibodies to salivary gland antigens (SGAs) from Culex tarsalis, the primary arbovirus vector in California. Chickens fed upon by Cx. tarsalis developed an antibody response that was proportional to the amount of exposure. Cross-reactivity between sera from Cx. tarsalis-exposed chickens and SGAs from Culex pipiens quinquefasciatus, Culex pipiens pipiens, Ochlerotatus melanimon, and Ochlerotatus sierrensis was likely due to shared SGAs among these species. This serologic assay for mosquito exposure could be used to evaluate the sensitivity of sentinel flocks for detecting arboviral activity.     

Preparation and use of monoclonal antibodies in the detection of Trichinella spiralis circulating antigen

In the present study, a trial was made to evaluate the monoclonal antibody produced as a tool in the detection of circulating antigen. For the preparation of monoclonal antibodies, four Balb/C mice were immunized with Trichinella spiralis larvae. Immunized spleen cells were prepared and a suitable mutant NS1 myeloma cell line was used for fusion. Eighty white swiss albino mice were orally infected with 500 L1 T. spiralis larvae. Their serum was collected at different periods i.e. 5, 11, 18 and 28 days post infection for the detection of circulating antigen which was done by the ELISA technique. Circulating antigen could not be detected on day 5 post infection, while on day 11 it was clearly identified; on day 18 it could be detected but at a lesser O.D. reading however the antigen disappeared completely on the 28th day. This study confirmed that monoclonal antibodies may be a valuable tool in the early diagnosis of trichinosis by the detection of specific antigens, even in small amounts whenever present in the circulation.     

Detection of Clostridium difficile toxins by enzyme immunoassay

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.     

Levels of the schistosome circulating anodic and cathodic antigens in serum of schistosomiasis patients from Brazil.

Serum levels of 2 schistosome circulating antigens, the circulating anodic antigen (CAA) and the circulating cathodic antigen (CAA), were determined in persons infected with Schistosoma mansoni in Brazil. Sensitive monoclonal antibody-based enzyme-linked immunosorbent assays were used to measure levels of the 2 antigens. The study group consisted of 38 individuals with intestinal schistosomiasis, and 20 persons with the hepatosplenic form of the disease. Age and intensity of infection were comparable for the 2 groups. CAA was detected in 65.5% of all patients' sera and CCA was found in the serum of 82.8% of all patients. CAA levels correlated well with the egg output, as determined by duplicate Kato-Katz smears; CCA was significantly positively correlated with egg output in patients with intestinal schistosomiasis only. Whereas no significant difference was found between CAA titre in patients with intestinal schistosomiasis and those with the hepatosplenic form, a significantly higher CCA titre was found in patients with hepatosplenomegaly compared to patients with intestinal schistosomiasis.     

Use of circulating cathodic antigen strips for the diagnosis of urinary schistosomiasis

Rapid diagnostic tests are needed for the implementation and monitoring of national schistosomiasis control programmes. The field applicability of the circulating cathodic antigen (CCA) urine reagent strip for the diagnosis of Schistosoma haematobium infection was evaluated among 265 pre- and primary schoolchildren aged 2-19 years in a rural area of Zimbabwe. The CCA strip was compared with egg detection before and six weeks after treatment with praziquantel. Pre-treatment prevalence (overall 40.4%) and intensity of infection, as determined by egg counts, increased with age. CCA and parasitological results were significantly correlated (P<0.001), although concordance was slight (kappa=0.21). Discordant results were mainly attributable to CCA-positive, egg-negative individuals. Correlations and levels of agreement improved significantly with age (P<0.001, kappa=0.40) and intensity of infection (P<0.001). Praziquantel treatment led to 'cure' in 90.9% and 70.5% of children as measured by the egg detection and CCA methods, respectively. An arbitrary gold standard was constructed that included both CCA and egg detection results. Using this standard, the sensitivities of the CCA test were 88.2% and 95.8%, respectively, for pre- and post-treatment results. The improved version that is field applicable now has an acceptable role in the field diagnosis of S. haematobium.     

Detection of Clostridium difficile toxins by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.     

Time-resolved immunofluorometric assay (TR-IFMA) for the detection of the schistosome circulating anodic antigen

We report the development of a time-resolved immunofluorometric assay (TR-IFMA) for the quantitative determination of the schistosome circulating anodic antigen (CAA). A mouse monoclonal antibody (line 120-1B10-A), recognizing a repetitive epitope on CAA, was used as both antigen-capture antibody and as Europium-labelled antigen-detecting antibody. The lower detection limit of the assay was 20 pg of trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, with a nearly linear measuring range from 20 pg to 130 ng AWA-TCA per ml. The TR-IFMA was compared with an enzyme-linked immunosorbent assay (ELISA), in which the same monoclonal antibody was used as antigen-capture antibody and as alkaline phosphatase-conjugated antibody. The lower detection limit of the TR-IFMA was tenfold lower than that of the ELISA, while the linear range of the TR-IFMA exceeded that of the ELISA one hundred-fold. Serum samples of 80 Burundese individuals infected with Schistosoma mansoni (egg counts ranging from 4 to 2583 eggs per gram of faeces) were tested in both assays. Antigen concentrations in serum of individuals infected with S. mansoni ranged from 0-500 ng AWA-TCA per ml. The correlation between antigen levels measured by TR-IFMA and ELISA was good: Spearman's p = 0.92. Whereas in the ELISA the samples had to be titrated, the wide linear range of the TR-IFMA allowed the assay to be performed at a single serum dilution, at which an exact estimation of the antigen concentration was possible.     

Determination of circulating filarial antigen using monoclonal antibody against Brugia malayi adult worm ES antigen with Dot-ELISA

A hybridoma cell line secreting monoclonal antibody (McAb) against Brugia malayi adult worm excretory-secretory antigen (AWES) was established. The identification of the Ig sub-classes showed that McAb AWES belongs to IgG. We developed a Dot-enzyme-linked immunosorbent assay (Dot-ELISA) to detect circulating parasite antigens in human lymphatic filariasis. 75 of 77 (97.4%) bancroftian microfilaremia cases showed positive reaction. 27 out of 40 (67.5%) microfilarial patients with hydroceles, chyluria, or elephantiasis, and 20% of sera from asymptomatic residents of filariasis-endemic areas evidently contained filarial antigens. 31 of sera from non-endemic area with ascaris infection were all negative. The minimal amount of antigens in pool normal sera adding AWES antigen was detectable at 6.3 pg/ml. The results suggested that Dot-ELISA is a sensitive, specific, cheap and simple agent for use in epidemiological field studies.     

Enzyme immunoassay for the detection of dengue IgG and IgM antibodies using infected mosquito cells as antigen

The detection of immunoglobulin (Ig)G and IgM antibodies to dengue 1 virus was studied by a simple enzyme immunoassay, in which infected cultured cells infected with dengue virus were used as antigen (EIA-ICC). Detection of anti-dengue 1 IgG by EIA-ICC was correlated with haemagglutination assays. EIA-ICC anti-dengue 1 IgM detection was less sensitive than IgM capture enzyme-linked immunosorbent assay. IgG and IgM responses in dengue 1 infection were studied by EIA-ICC, using sera collected at different intervals after onset of illness: IgM and IgG appeared on the 4th day of disease; the highest IgM mean titres were detected on the 7th day and IgM was not detected in sera obtained after the 60th day; the highest mean titres of anti-dengue 1 IgG were seen in sera obtained between 22 and 30 d after onset of illness. EIA-ICCs for 6 flaviviruses and 1 alphavirus were conducted with sera from patients infected with dengue 1, and primary and secondary infections of other flaviviruses. The results showed that anti-dengue 1 IgG detection was sensitive, and the antibodies were cross-reactive among the flaviviruses. Anti-dengue 1 IgM detected in dengue 1 patients was mostly type specific. The pattern of secondary dengue infection, i.e. the presence of IgG and a low titre or absence of IgM antibodies, was observed in the sera of 6 patients obtained in the first week after onset of illness. EIA-ICC is useful for dengue diagnosis, surveillance and sero-epidemiological studies.     

Detection of circulating anodic antigen by ELISA for seroepidemiology of schistosomiasis mansoni

Sera of individuals from Burundi excreting eggs of Schistosoma mansoni (prevalence 35%; 178 subjects) and of similar individuals from Maniema, Zaire (prevalence 95%; 99 subjects), and of 159 Dutch and 81 Zairean non-infected controls, were screened by enzyme-linked immunosorbent assay for the presence of schistosome circulating anodic antigen (CAA). No false positive results were obtained. The sensitivity of the test was 75% in Burundi and 93% in Zaire, a significant difference (P less than 0.05). However, in matched egg output classes the test results did not differ significantly; 60% and 67%, respectively, of those excreting 1-100 eggs per gram of faeces (epg), 86% and 100% of those excreting 101-400 epg, and 100% of those excreting over 400 epg were detected. The efficiency of the assay was 91% in Burundi and 93% in Zaire. The Spearman rank coefficient of correlation between antigen titre and egg output (determined by 3 consecutive Kato egg counts) was 0.61 in Burundi and 0.82 in Zaire. The sensitivity of the test compared well with a single egg count. In addition, preliminary data showed that occasionally CAA was detectable in serum of individuals not excreting schistosome eggs. As CAA is found only in the presence of living worms, such cases reflect active infections.     

Detection of antimicrofilarial ES antigen-antibody in immune complexes in Bancroftian filariasis by enzyme immunoassay

An enzyme immunoassay using penicillinase conjugated to Wuchereria bancrofti microfilarial ES antigen has been developed to detect specific antibody in circulating immune complexes in Bancroftian filariasis. Immune complexes were prepared by 3% polyethylene glycol (PEG) precipitation. 44 sera belonging to different groups were tested. 16 of 19 clinical filarial and two of 16 endemic normal sera but none of the non-endemic normal sera showed the presence of antimicrofilarial ES antigen-antibody in immune complexes.     

Determination of Legionella antigens using monoclonal antibodies

The detection of antigens is the most important tool for rapid diagnosis of Legionellosis. 34 Legionella (L.) spp. with 51 serogroups have been identified from several sources. According to the antigenic diversity, it is necessary to select monoclonal antibodies (mab) adequate to diagnostic purposes. Mab with specificity to genus, species and serogroups were discussed. L. pneumophila accounts for 70 to 80% of all cases of Legionelloses. In this study self-made mab to L. pneumophila are presented that demonstrate these bacteria in clinical materials from respiratory tract using immunofluorescent tests and by detection of soluble antigens in pleura fluids and urine specimens using enzyme immunoassay.     

Immunoscintigraphy of choroid melanoma using monoclonal antibodies

A prospective pilot study on immunoscintigraphy with monoclonal antibody fragments against cutaneous melanoma (MoAb 225.28S) was performed in 17 patients with a clinical diagnosis of choroidal melanoma. Monoclonal antibodies against melanoma associated antigen were labelled with 740 mBq (20 MCi) of the radionuclide 99mTc and injected intravenously. Images were made with a gamma camera 6 hours after injection. With a double pinhole collimator radioactivity was counted thrice in both eyes 6 hours after injection. In 6 out of 17 patients (35.3%) the melanoma could be imaged with the gamma camera. With the double pinhole collimator a significantly higher activity was measured in the melanomatous eye in 13 out of 17 patients (76.5%). In four patients a false negative result was obtained. Considering these results immunoscintigraphy may be valuable in ocular melanoma diagnostics but its specific value still has to be assessed.     

Human neurocysticercosis: comparison of enzyme immunoassay capture techniques based on monoclonal and polyclonal antibodies for the detection of parasite products in cerebrospinal fluid

Current diagnosis of neurocysticercosis relies mostly on computerized tomography and nuclear magnetic resonance, with detection of antibodies being confirmatory rather than decisive. An assay which detects parasite products in cerebrospinal fluid would conclusively demonstrate a current infection and could be important when decisions regarding treatment must be made. Cerebrospinal fluid from patients with neurocysticercosis was used in 4 enzyme immunoassay capture tests designed to detect parasite products. Of the systems tested, one, based on the use of a monoclonal antibody reactive with a surface and secretion component of the metacestode, was particularly promising, giving a sensitivity of 72%. The assay has the double advantage of a very low background and a proved specificity for the products of living cysticerci. The other 3 systems (monoclonal anti-vesicular fluid antibody, polyclonal antibody against a saline extract and polyclonal anti-antigen B antibody) were less sensitive. Results with the anti-antigen B system support the proposal that products of low immunogenicity are the most appropriate targets for the serological detection of the parasite.