Deglycosylation of Toxocara excretory–secretory antigens improves the specificity of the serodiagnosis for human toxocariasis

Serodiagnosis of human toxocariasis is difficult in tropical areas where other helminthiasis are endemic. Many studies have shown that glycans from helminths may be the responsible for cross‐reactions in the immunoassays. In this study, we have evaluated the deglycosylation of the Toxocara canis excretory–secretory (TES) antigens for the detection of IgG antibodies using a panel of 228 serum samples (58 patients with toxocariasis, 75 patients with other helminth infections and 95 healthy individuals) by ELISA and Western blot assays. Our results showed that the deglycosylation of TES antigens resulted in a single fraction of 26 kDa (dTES) and was able to detect IgG antibodies with a sensitivity and specificity of 100% in both above‐mentioned assays. The rate of cross‐reactions, observed in ELISA with TES (13·3%), was significantly reduced (5·3%) when the dTES antigens were used. Likewise, the cross‐reactivity observed with the fractions of 32, 55 and 70 kDa of the TES antigens was totally eliminated when the dTES were used in the Western blot. All these results showed that the deglycosylation of the TES antigens really improves the specificity of the serodiagnosis of human toxocariasis in endemic areas for helminth infections.     

The production and comparative evaluation of native and recombinant antigens for the fast serodiagnosis of cystic echinococcosis with dot immunogold filtration assay

Clinical diagnosis and post‐surgery assessment of cystic echinococcosis depend on laboratory serodiagnosis and ultrasound examinations. This study aims to produce the recombinant antigen (rAgB) and compare its diagnostic effect with natural antigens (crude fluid antigen, protoscolex antigen). After rAgB, crude fluid antigen, protoscolex antigen were produced, and the diagnostic accuracy was evaluated with dot immunogold filtration assay (DIGFA) by the sera from the following groups: surgically confirmed cystic echinococcosis patients (n = 113), alveolar echinococcosis patients (n = 46), other parasitic diseases (n = 49), nonparasitic hepatic diseases (n = 63) and healthy people (n = 121). In diagnosing cystic echinococcosis, the sensitivity of recombinant AgB was 77·9% and the specificity was 98·3%. The crude fluid antigen B showed a sensitivity of 92·9% and specificity of 81·0%. The protoscolex antigen had sensitivity of 87·6% and specificity of 90·9%. The recombinant AgB indicates the advantage of no cross‐reaction with other parasite diseases or nonparasite hepatic diseases. Recombinant antigen B can improve the specificity but decrease the sensitivity. The combination of native and recombinant antigens will improve the overall performance of serodiagnosis of cystic echinococcosis.     

Serodiagnosis of neuroborreliosis: Comparison of reliability of three confirmatory assays

Summary The sensitivity and specificity of three confirmatory assays for the serodiagnosis of neuroborreliosis were investigated. Samples from 96 patients with proven neuroborreliosis, 80 healthy volunteers, 20 patients with neurosyphilis and 20 patients with recent infections with Epstein-Barr virus (EBV) were tested for borrelial antibodies by immunoblotting,Borrelia burgdorferi sensu lato sonicate EIA following pre-absorption of cross-reactive antibodies (Abs-EIA) and by a so-called RECO-EIA using the following recombinant borrelial proteins as antigens: a 14 kDa-internal flagellin fragment, the outer surface protein C (23 kDa) and the high molecular mass protein p83 (83 kDa). the immunoblots were evaluated according to the criteria published byEngstr?m et al. andHauser et al. An evaluation of IgM and/or IgG antibodies revealed a considerably higher sensitivity for the RECO-EIA (94%) compared to the Abs-EIA (82%, P<0.0001). Evaluation of the immunoblot according to the criteria ofHauser was significantly more sensitive than according to the criteria ofEngstr?m (89 vs 51%, P=0.0003). A higher sensitivity was demonstrated for IgM (54 vs 22%) and IgG antibodies (64 vs 24%). When both findings from RECO-EIA and immunoblotting were considered, positive findings in the first step assay (sonicate EIA without pre-absorption) were confirmed in 97% of patients. When samples were tested for IgM antibodies, the specificities of the three confirmatory assays did not differ significantly, but in the case of IgG antibodies, the immunoblot (Hauser: P=0.013;Engstr?m: P=0.004) and the RECO-EIA (P=0.02), were more specific than the Abs-EIA. It is concluded that the immunoblot (evaluated according toHauser) and the RECO-EIA are both suitable as confirmatory assays in the serological diagnosis of neuroborreliosis. Monoclonal antibodies are mandatory tools in the evaluation of the immunoblot.     

Evaluation of a di‐O‐methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis

Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme‐linked immunosorbent assay (ELISA) using Toxocara larvae excretory–secretory (TES) antigens, but its production is a laborious and time‐consuming process being also limited by the availability of adult females of T. canis as source for ova to obtain larvae. Chemical synthesis of the di‐O‐methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n = 60), patients with other helminth infections (n = 75) and healthy individuals (n = 94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross‐reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis.     

弓形虫速殖子表膜蛋白的组分和抗原性分析

目的试验分析弓形虫速殖子表膜蛋白的组分及其抗原性。方法用聚丙烯酰胺凝胶电泳分析弓形虫速殖子表膜蛋白的组分和用弓形虫病人阳性血清及兔抗弓形虫表膜抗原血清识别硝酸纤维膜载体上的速殖子表膜抗原的免疫反应靶位。结果弓形虫速殖子表膜抗原的独特蛋白区带分子为16和17kDa,弓形虫病人特异性抗血清识别的速殖子表膜抗原的免疫反应位点为16kDa,免抗血清识别出64,35,32,26,16kDa等8个位点。结论弓形虫速殖子表膜蛋白存在特异的抗原决定簇和可被特异性抗体识别的受体,证明速殖子表膜蛋白具有较好的抗原性,此对于弓形虫病的免疫学诊断具有重要的意义。     

Evaluation of recombinant antigens of Trypanosoma cruzi to diagnose infection in naturally infected dogs from Chaco region,Argentina

Dogs are considered the main mammal reservoir of Trypanosoma cruzi in domiciliary environments. Consequently, accurate detection of T. cruzi infection in canine populations is epidemiologically relevant. Here, we analysed the utility of the T. cruzi recombinant antigens FRA, SAPA, CP1, Ag1 and a SAPA/TSSA VI mixture, in an ELISA format. We used a positive control group of sera obtained from 38 dogs from the Chaco region in Argentina with positive homogenate‐ELISA reaction, all of them also positive by xenodiagnosis and/or PCR. The negative group included 19 dogs from a nonendemic area. Sensitivity, specificity, area under the curve (AUC) of the receiver operating charactheristic (ROC) curve and Kappa index were obtained to compare the diagnostic efficiency of the tests. The SAPA/TSSA VI had the highest performance, with a sensitivity of 94·7% and an AUC ROC of 0·99 that indicates high accuracy. Among individual antigens, SAPA‐ELISA yielded the highest sensitivity (86·8%) and AUC ROC (0·96), whereas FRA‐ELISA was the least efficient test (sensitivity = 36·8%; AUC ROC = 0·53). Our results showed that the use of SAPA/TSSA VI in ELISAs could be a useful tool to study dogs naturally infected with T. cruzi in endemic areas.     

Immunoglobulin class and subclass distribution of eye muscle and fibroblast antibodies in patients with thyroid-associated ophthalmopathy

OBJECTIVES The investigation of the antibody response in thyroid-associated ophthalmopathy (TAO) using different antigens and assays has given inconsistent results. We have analysed antibodies against eye muscle and control antigens in a large group of TAO patients to assess whether specific eye muscle antibodies exist in TAO. We have also evaluated the presence of IgA and IgM class antibodies and examined IgG subclass distribution. DESIGN Sera were obtained from all patients (TAO, Graves' disease without ophthalmopathy and Hashimoto's thyroiditis) within one year of diagnosis. Sera were also collected from healthy controls, with no family history of autoimmune thyroid disease. PATIENTS Thirty-eight patients had Graves' disease with Grade III or greater TAO; 15 patients had Graves' disease without ophthalmopathy and nine had Hashimoto's thyroiditis without any eye signs. The control group consisted of 14 subjects. MEASUREMENTS Antibodies against porcine eye and skeletal muscle, human eye (membrane and soluble antigen) and skeletal muscle, human thyroid microsomal and thyroglobulin antigens and dermal and orbital fibroblast antigens were assessed using ELISA. Antibody isotypes and IgG subclasses were studied for porcine and human eye muscle antibodies. Eye muscle (porcine and human) and orbital fibroblast antibodies were further analysed by immunoblotting. RESULTS There were no significant differences in the ability of either IgG or IgA in sera from the different groups to bind porcine and human eye muscle antigens. There was a significant correlation (P < 0·0001) between the binding to porcine eye muscle and skeletal muscle antigens (for both IgG and IgA). There was no difference between sera from TAO patients and control subjects in their binding to eye muscle fibroblasts for both IgG and IgA antibodies. However, IgA antibody activity against dermal fibroblasts differed significantly between TAO patients and controls (P <0·05). By immunoblotting, the frequency of IgA antibodies recognizing 21 kDa (40% of patients) and 62 kDa (52%) bands in porcine eye muscle blots and 20, 24 and 38 kDa bands in blots of human eye muscle (soluble) antigen differed significantly between patients with TAO and controls (P <0·05 in all cases). IgG antibodies recognizing 80 and 92 kDa bands in blots of the subcellular membrane antigen prepared from orbital fibroblasts were found more frequently in patients with TAO compared with controls (P <0·05 in both cases). CONCLUSIONS We found no evidence that eye muscle membrane or fibroblast antibodies are present in a significant proportion of TAO patients, using ELISAs based on antigens prepared from several sources. We have also failed to demonstrate the presence of previously described specific, TAO-associated antibodies, including those directed against a 64 kDa protein in eye muscle and a 23 kDa protein in fibroblasts. IgA class antibodies reactive with orbital components appeared to be more strongly associated with TAO than those of the IgG class, though even this relationship is weak. These results suggest that antibodies are of secondary importance in the pathogenesis of TAO, which is most likely a T cell-mediated disorder.     

Laboratory detection of strongyloidiasis: IgG‐, IgG4‐ and IgE‐ELISAs and cross‐reactivity with lymphatic filariasis

Enzyme‐linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG₄ and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG₄, IgG, IgE and IgG (IVD) assays were 76·9%, 84·6%, 7·7% and 84·6%, respectively, while the specificities were 92·7%, 81·8%, 100% and 83·6%, respectively. If filariasis samples were excluded, the specificities of the IgG₄‐ELISA and both IgG‐ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG‐ and IgG₄‐ELISAs (r = 0·4828; P = 0·0125). IgG‐ and IgG‐ (IVD) ELISAs (r = 0·309) were positively correlated, but was not significant (P = 0·124). Meanwhile there was no correlation between IgG₄‐ and IgG‐ (IVD) ELISAs (r = 0·0042; P = 0·8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG₄ and the optical densities of anti‐Strongyloides IgG₄‐ELISA (r = 0·4544, P = 0·0294). In conclusion, the detection of both anti‐Strongyloides IgG₄ and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.     

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.     

重组抗原在弓形虫病诊断中的应用

刚地弓形虫感染危害严重,研究敏感性高和特异性好的弓形虫感染检测方法对于弓形虫病的诊断至关重要。弓形虫病的诊断一般依赖于检测患者血清中弓形虫特异性IgM和IgG。目前,商业试剂盒大多采用天然抗原,其检测精确性较差。重组抗原已经被发展用来诊断弓形虫病,且具有潜在的应用价值。本综述主要介绍重组抗原在弓形虫病诊断中的应用。     

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38 kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38 kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38 kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.     

肺孢子虫病实验与临床研究 Ⅱ．免疫印迹试验检测人血清肺孢子虫抗体

采用ＳＤＳ－ＰＡＧＥ分析鼠源卡氏肺孢子虫包囊可溶性抗原呈现２０条以上多肽区带，主带分子量分别为＞１００、８５－９４、６７、５２和３４ｋＤａ。ＥＩＴＢ检测表明重庆地区健康人血清中存在识别１１０、１０５、８５、６７、５２和４６ｋＤａ的ＩｇＧ型抗体，抗体阳性率为５６．７％（５９／１０４）。其中８５ｋＤａ抗原能与抗人源肺孢子虫单克隆抗体２Ｇ２起反应。健康人血清抗８５ｋＤａ抗原的抗体阳性率为３７．５％（３９／１０４），但ＩｇＭ型抗体均为阴性。另外检测７例确诊的卡氏肺孢子虫病患者，４例血清ＩｇＧ型抗体阳性，其中３例抗８５ｋＤａＩｇＧ型抗体阳性。     

Western immunoblot analysis using a ten leptospira serovars combined antigen for serodiagnosis of leptospirosis

Leptospirosis is a major zoonotic disease throughout the world. There are unavailable accuracy diagnostic methods for the acute phase of infection. To demonstrate the advantage of Western immunoblot, a mixed leptospira serovars antigen for the serodiagnosis of leptospirosis was employed. SDS-PAGE and Western immunoblot was performed using a 10 mixed leptospira serovars antigen and stained with 16 reference rabbit anti-leptospirosis antibodies. The result showed different immunoreactive band patterns for each reference serum. The bands with molecular weights of 15-20, 23-24, 41 and 45 kDa were commonly found (88% to 100% of the 16 reference sera). Using combined leptospira antigens in a Western immunoblot technique is an alternative and practical strategy for a more sensitive leptospirosis serodiagnosis.     

弓形虫速殖子抗原组分分析及其免疫反应性的比较研究

应用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、酶联免疫印渍试验(ELIB)和酶联免疫吸附试验(ELISA),对四种不同地理株弓形虫-CN、RH、BH、HH速殖子细胞质、细胞膜和代谢抗原组分及株间抗原差异、免疫反应性进行比较研究。ELIB实验结果:RH株高免兔血清与四株细胞质抗原反应,分别出现10、7、4、6条显色的反应区带,其中在35～38kD处都出现极明显的反应区带;与四株细胞膜抗原反应,分别出现5、6、6、9条显色反应区带,其中在31～35kD处都出现极明显的反应区带;与四株代谢抗原反应,分别出现8、7、6、7条反应区带,其中在50kD处、100～108kD处都有明显反应区带。实验结果提示,高免兔血清与四株12种抗原反应结果所出现的区带存在不同程度差异。ELISA检测免疫比值(P/N)大小比较结果表明,无论是细胞质、细胞膜或代谢抗原,在检测人弓形虫抗体阳性血清时,HH、BH较CN、RH的免疫比值高,株间抗原免疫反应似存在一定差异。     

Persistence of serum antibodies to Borrelia burgdorferi in patients treated for Lyme disease.

To determine if antibodies to Borrelia burgdorferi persist after antibiotic treatment, we recalled 32 patients with Lyme disease from a primary care practice a mean of 16 months after treatment and analyzed initial and follow-up serum samples by ELISA and immunoblot assays. Of the eight patients whose initial serum specimens were positive for IgM antibody by ELISA, three had positive titers of IgM antibody at follow-up; of the 23 patients whose initial serum specimens were positive for IgG antibody by ELISA, 19 had positive titers of IgG at follow-up. Of the five patients whose initial serum specimens were positive for IgM antibody by immunoblot, two had positive titers of IgM antibody at follow-up; of the 30 patients whose initial serum specimens were positive for IgG antibody by immunoblot, 29 had positive titers of IgG antibody at follow-up. The bands on the IgG immunoblot remained remarkably constant during the period from analysis of the initial specimen to that of the follow-up specimen. Nine of the 32 patients had persistent or recurrent symptoms, and ELISA and immunoblot were not helpful for identifying these nine patients.     

旋毛虫成虫三种抗原SDS-PAGE及Western-blot比较研究

目的研究大理猪源旋毛虫(Trichinella spiralis)成虫虫体可溶性抗原、表面抗原和排泄分泌抗原3种抗原的蛋白组分及其之间的差异,比较分析其免疫反应性。方法以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白印迹对成虫3种抗原进行蛋白组分及其免疫反应性分析。结果十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果:虫体可溶性抗原显示29条蛋白带,分子量范围在112kDa~10kDa之间,其中主带13条;表面抗原显示4条主要蛋白带,分子量分别为50、48、40、34kDa;排泄分泌抗原显示17条蛋白带,分子量范围在120~14kDa之间,其中主带6条,分子量分别为120、64、43、40、35、33kDa。蛋白印迹结果:虫体可溶性抗原出现13条反应条带,其中分子量为81、40、37、33、30kDa的条带着色明显;表面抗原出现4条反应条带,其分子量分别为50、48、40、34kDa;排泄分泌抗原显示7条反应带,其中43、40、27、18kDa着色明显。结论旋毛虫成虫虫体可溶性抗原与排泄分泌抗原较表面抗原蛋白组分复杂,3者主带部分属低分子量区;免疫反应性的强度表面抗原和排泄分泌抗原高于虫体可溶性抗原,说明前两种抗原是旋毛虫成虫刺激机体产生免疫应答的主要靶抗原。     

旋毛虫、卫氏并殖吸虫及华支睾吸虫之间共同抗原的研究

目的鉴定旋毛虫、卫氏并殖吸虫及华支睾吸虫之间的共同抗原,避免血清学诊断这3种寄生虫病时的交叉反应。方法应用十二烷基硫酸钠-聚丙烯酸胺凝胶电泳(SDS-PAGE)和免疫印渍(Western blot)对旋毛虫肌幼虫、卫氏并殖吸虫成虫及华支睾吸虫成虫可溶性抗原中的蛋白组分进行研究。结果旋毛虫肌幼虫、卫氏并殖吸虫成虫及华文睾吸虫成虫3者相同蛋白带的分子量为108、65、53、43、42、31、25、16 kDa。免疫印渍结果表明,旋毛虫和卫氏并殖吸虫抗原中分子量为 65、58、53kDa的蛋白带均与旋毛虫感染的大鼠、小鼠及患者血清、肺吸虫感染的大鼠和患者血清发生反应;旋毛虫和华支睾吸虫抗原中分子量为108kDa的蛋白带均与旋毛虫感染的大鼠、小鼠及患者血清、肝吸虫病患者血清发生反应;而3种可溶性抗原中的53kDa与本实验中所有寄生虫感染的动物和患者均有交叉反应。结论 65、58、53 kDa蛋白为旋毛虫和卫氏并殖吸虫的共同抗原,108kDa蛋白为旋毛虫和华支睾吸虫的共同抗原,而53 kDa蛋白为这3种寄生虫的共同抗原。     

Humoral immune response against 38‐ and 16‐kDa mycobacterial antigens in childhood tuberculosis

Several enzyme‐linked immunosorbent assays (ELISAs) based on mycobacterial antigens have been tried for the rapid diagnosis of tuberculosis (TB). In this study, the value of the 16 and 38‐kDa mycobacterial antigens in the diagnosis of TB was investigated in pediatric patients in Izmir, Turkey in whom they were found using clinical and/or bacteriological methods. A commercial ELISA kit was used for measuring IgG against 38 and 16‐kDa recombinant antigens. The humoral immune response was analyzed in a group of 32 TB patients (24 pulmonary, 3 lymphadenitis and 2 pleuritis, 2 meningitis and a disseminated TB) and in control groups consisting of 20 healthy children and 20 pulmonary diseases other than TB cases. The sensitivity, specificity, positive predictive value, and the negative predictive value of the test were found to be 25%, 90%, 66.7%, and 60%, respectively, in the TB cases. The ELISA test shows very good specificity, but low level of sensitivity and negative predict value. It was thought that it might be used in combination with other methods to increase diagnostic accuracy, especially for culture‐negative TB pediatric cases, which are difficult to diagnose. Pediatr Pulmonol. 2009; 44:839–844. © 2009 Wiley‐Liss, Inc.     

田鼠巴贝虫可溶性抗原组分分析及其初步应用

目的 分析田鼠巴贝虫可溶性抗原,寻找可用于免疫诊断的有效抗原组分.方法 用田鼠巴贝虫感染BALB/c小鼠,待虫血症达高峰期时,收集田鼠巴贝虫虫体;采用超声法制备可溶性粗抗原（soluble babesia antigens,SBA）并包板,ELISA检测血清特异性IgG,评价SBA的免疫反应性、交叉反应性和特异性;以SDS-PAGE电泳分析SBA组分,并进行Western Blot,分析其与感染鼠血清的反应性.结果 SBA-ELISA法可检测早期感染（7 d）小鼠血清,且与恶性疟、间日疟弓形虫病阳性血清无交叉反应,显示出其较好的诊断敏感性和较高的特异性.通过SDS-PAGE分析,获得5条分子质量为72、66、60、53、43 kDa的主蛋白带和介于14.4～116 kDa的7条次带.经Western Blot分析,SBA有15个抗原组分能被阳性小鼠血清识别,以72、53、43、39、30 kDa蛋白组分反应较强.结论 以SBA建立的间接ELISA可用于巴贝虫感染的有效筛查工具;田鼠巴贝虫可溶性抗原组分中72、53、43、39、30KDa为比较理想的抗原组分.     

Application of Toxocara canis excretory-secretory antigens and IgG subclass antibodies (IgG1-4) in serodiagnostic assays of human toxocariasis

A major problem in the serodiagnosis of human toxocariasis in tropical countries is cross-reaction with antibodies to other helminthic diseases and a lack of sensitivity. The majority of tests currently available use total IgG and, in this study, the use of peroxidase-conjugated anti-human IgG subclass antibodies (IgG1-4) was compared with total IgG for the diagnosis of human toxocariasis by using Toxocara excretory–secretory (TES) antigens in an enzyme-linked immunosorbent assay (ELISA) format. All four IgG subclass antibodies gave approximately 10-fold increases in optical density (OD) values for 50 toxocariasis patients compared to 29 healthy normals; this was significantly greater than the approximate doubling of OD values seen in the total IgG-ELISA format. IgG2 gave by far the greatest sensitivity (values: IgG, 50%; IgG1, 60%; IgG2, 98%; IgG3, 78%; IgG4, 64%). Significant cross-reactivity using all IgG subclasses in the TES ELISA was seen with 141 serum samples from patients with 10 other helminthic infections. However, IgG3 gave the best specificity (values: IgG, 73%; IgG1, 76%; IgG2, 71%; IgG3, 81%; IgG4, 71%). Thus, of the IgG subclass antibodies, IgG2 appeared best and employing this subclass can improve the serodiagnosis of human toxocariasis since it recognises carbohydrate epitopes of TES antigens.