抗鸡IgM(μ链)单克隆抗体杂交瘤细胞株的建立与鉴定

以绵羊红血球免疫鸡,获得富含IgM的鸡血清。以提纯鸡血清IgM免疫BALB/C小鼠,取免疫鼠脾细胞与SP 2/0骨髓瘤细胞融合,用间接ELISA方法进行筛选,共获得7株稳定分泌特异性抗鸡IgM杂交瘤细胞)TM18、TM21、TM25、TM34、TM57、TM62、TM65)。杂交瘤培养上清的ELISA效价为10~3～10~5,腹水型单克隆抗体的ELISA效价为10~5～10~7。7株单抗中TM57、TM65为小鼠IgM类,其余5株均为小鼠IgG_1亚类。间接ELISA和夹心ELISA试验表明,所有7株单抗只与鸡IgM反应,而不与鸡IgG和鸡IgA反应。7株单抗均能抑制鸡IgM对其特异抗原绵羊红血球的血凝作用。血凝抑制价为1∶2~3～2~7,7株单抗中只有TM65能在琼脂扩散试验中与鸡IgM出现沉淀线.     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

ELISA检测鸡新城疫病毒特异性IgM抗体的研究

以新城疫病毒单克隆抗体包被板,用10％小牛血清-PBS封闭后,捕获尿囊液中的新城疫病毒作固相抗原.在此板上应用酶标抗鸡IgM单克隆抗体进行间接ELISA试验检侧鸡血清中新城疫病毒的特异性IgM抗体.试验证明该方法特异性强、敏惑性高.兔抗新城疫病毒阳性血清可特异性阻断反应,将新城疫病IgM阳性血清用2-ME处理可使ELISA反应呈阴性,与禽源多杀性巴氏杆菌鸡IgM阳性血清、鸡传染性支气管炎病毒IgM阳性血清无交叉反应.该试验可检测到La Sota免疫后3天鸡血清中的特异性IgM,对鸡新城疫病毒IgM阳性血清的检测效价可达1:320以上,并可检测到临床新城疫病鸡血清中的特异性IgM抗体.     

Development and Primary Application of Hybridoma Cell Lines Secreting Monocional Antibodies Against LPS Antigen of B.Melitensis

研制抗羊种布鲁菌脂多糖抗原的单克隆抗体,并应用其建立检测布病的双夹心ELISA方法.本研究采用热酚水法提纯羊种布鲁菌(16M菌株)的脂多糖抗原,并经SDS-PAGE鉴定.用脂多糖和灭活的羊种布鲁菌16M作为免疫抗原,交替免疫6～8周龄BAIB/c雌鼠,第1次免疫用羊种布鲁菌标准菌16M全菌加等量弗氏完全佐剂;第2次免疫用脂多糖加等量弗氏不完全佐剂.将脂多糖作为包被抗原建立间接ELISA方法,筛选针对抗羊种布鲁菌(16M菌株)脂多糖的单克隆抗体杂交瘤细胞株.筛选出3株能稳定分泌抗羊种布鲁菌脂多糖单克隆抗体的杂交瘤细胞株,分别命名为5H3、6B8和3H7,细胞培养上清的ELISA效价在1:1 000～1:5 000,小鼠腹水单克隆抗体ELISA效价在1:10 000～1:160 000;抗体亚类鉴定表明:5H3、6B8属于IgM亚类,3H7属于IgG3亚类;特异性试验结果显示:3株杂交瘤细胞分泌的抗体不与大肠杆菌O157裂解抗原、鸡白痢沙门氏菌裂解抗原、鸭源鸡杆菌脂多糖抗原以及福氏志贺菌裂解抗原反应,仅与灭活的羊种布鲁菌(16M)发生反应.布鲁菌虎红平板凝集试验和试管凝集试验检测结果显示,获得的单抗可与标准检测抗原形成明显的颗粒凝集物和伞状凝集物.利用所建立的单克隆抗体细胞株,建立了一种检测布鲁菌的双夹心间接ELISA方法,并进行了特异性和敏感性检测.对模拟样品和临床样品进行检测,准确性均很高.     

猪囊虫TS21抗原DNA接种小鼠的免疫应答

将40只BALB／c小鼠随机分为2组，A组以VR1020免疫作为对照，B组以TS2l抗原基因的真核表达型质粒VTS2l免疫。用ELISA检测免疫小鼠IgG总量和特异性抗体水平，MTT比色法检测小鼠脾淋巴细胞伴刀豆蛋白A(ConA)刺激的增殖反应及IL—2的谤生活性，常规法检测外周血免疫细胞数量的动态变化。结果显示，VTS2l免疫小鼠血清的IgG含量和特异性抗体效价显著高于对照组小鼠；免疫小鼠脾淋巴细胞ConA刺激增殖反应和IL—2诱生活性均比对照组小鼠显著增强；免疫小鼠的淋巴细胞、巨噬细胞等免疫细胞的数量也显著超过对照组。免疫小鼠的细胞和体液免疫反应显著增强，表明VTS2l具有很强的免疫激活作用，有进一步研制开发成为猪囊虫病DNA疫苗的潜力。     

犬GM-CSF基因对犬细小病毒VP2 DNA疫苗的免疫增强作用

犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白.利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应.为进一步提高VP2 DNA疫苗的免疫活性,本实验利用犬粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因作为生物佐剂研究其对犬细小病毒VP2 DNA疫苗的免疫增强作用.首先通过RT-PCR方法从犬淋巴细胞中扩增GM-CSF基因,并将其插入到pcDNA3.1栽体上,分别构建该基因的两个分泌型真核表达载体,即非融合表达载体pcDNA-cGMCSF和与Myc His融合的表达栽体pcDNA-cGMCSF/MH.用pcDNA-cGMCSF/MH载体转染HEK293T细胞以确定GM-CSF基因能否在真核细胞中进行分泌表达.然后用本室构建的VP2基因表达栽体单免疫小鼠,用VP2表达载体与pcDNA-cGMCSF共免疫小鼠(pcDNA3.1空载体作为阴性对照).免疫后用ELISA方法检测不同时间小鼠血清的抗体水平.用MTT法检测小鼠免疫后35 d时淋巴细胞的增殖活性,同时用ELISA试剂盒检测小鼠淋巴细胞γ干扰素的表达水平.结果表明,本试验构建的表达载体能够介导重组GM-CSF在真核细胞中进行分泌表达.免疫实验表明,利用GM-CSF基因与VP2基因共免疫小鼠,抗体的水平明显高于VP2基因单免疫组(P＜0.01).共免疫组小鼠淋巴细胞的刺激指数和γ干扰素的表达水平均明显高于单免疫组(P＜0.05).由此可见,GM-CSF表达载体可明显提高CPV VP2 DNA疫苗的免疫应答水平.     

沙门氏菌fimY基因的原核表达及禽沙门氏菌抗体检测ELISA研究

为研发禽沙门氏菌抗体检测方法,采用大肠杆菌系统表达沙门氏菌属保守的菌毛fimY基因,纯化的目的蛋白经Western blot分析,能与免疫鸡血清和感染鸡血清产生特异性反应条带,证明该重组蛋白既有反应原性也有免疫原性。以此纯化蛋白为抗原建立了间接ELISA(fimY-ELISA)。通过检验已知沙门氏菌鸡源血清、相关非沙门氏菌鸡源血清对fimY-ELISA进行评价,发现其在敏感性方面高于快速平板凝集试验(RPA)和自制的脂多糖抗原ELISA(LPS-ELISA)、肠炎沙门氏菌抗原ELISA(SE-ELISA),但在特异性方面不如LPS抗原ELISA;在与RPA的符合率方面与LPS-ELISA、SE-ELISA基本一致。     

细菌CpG DNA对鸡接种禽流感病毒H9亚型灭活苗的免疫增强作用

为了探讨细菌核酸对 H9亚型禽流感病毒灭活疫苗的免疫增强作用 ,将 1 5日龄蛋用公鸡 1 2 0只随机分为 4组 ,即空白组、抗原组、细菌核酸佐剂组及白油佐剂组 ,每组 30只。于 2 8日龄时 ,细菌核酸佐剂组以灭活 H 9亚型禽流感病毒作为抗原 ,大肠杆菌牦牛株核酸作为佐剂 ,免疫接种于试验鸡 ,用 XTT法检测 T、B淋巴细胞增殖反应 ,并用血凝与血凝抑制法检测血清抗体水平。结果显示 ,在细菌核酸佐剂组 ,鸡的血凝抑制抗体水平和 T、B淋巴细胞增殖反应均显著高于抗原单独免疫组 (P<0 .0 5 ) ,而免疫组均显著高于空白组 (P<0 .0 5 ) ;细菌核酸佐剂组的抗体滴度峰值略高于白油佐剂组 (P>0 .0 5 ) ,但 T、B淋巴细胞增殖指数峰值显著高于白油佐剂组 (P<0 .0 1 )。结论认为 ,大肠杆菌牦牛株核酸可显著增强免疫鸡对禽流感病毒 H9亚型灭活疫苗的免疫反应 ,其免疫增强效果略高于常规油佐剂     

稳定分泌抗羊种布鲁菌脂多糖单克隆抗体细胞株的筛选与应用

研制抗羊种布鲁菌脂多糖抗原的单克隆抗体,并应用其建立检测布病的双夹心ELISA方法。本研究采用热酚水法提纯羊种布鲁菌(16M菌株)的脂多糖抗原,并经SDS-PAGE鉴定。用脂多糖和灭活的羊种布鲁菌16M作为免疫抗原,交替免疫6～8周龄BALB/c雌鼠,第1次免疫用羊种布鲁菌标准菌16M全菌加等量弗氏完全佐剂;第2次免疫用脂多糖加等量弗氏不完全佐剂。将脂多糖作为包被抗原建立间接ELISA方法,筛选针对抗羊种布鲁菌(16M菌株)脂多糖的单克隆抗体杂交瘤细胞株。筛选出3株能稳定分泌抗羊种布鲁菌脂多糖单克隆抗体的杂交瘤细胞株,分别命名为5H3、6B8和3H7,细胞培养上清的ELISA效价在1∶1 000～1∶5 000,小鼠腹水单克隆抗体ELISA效价在1∶10 000～1∶160 000;抗体亚类鉴定表明:5H3、6B8属于IgM亚类,3H7属于IgG3亚类;特异性试验结果显示:3株杂交瘤细胞分泌的抗体不与大肠杆菌O157裂解抗原、鸡白痢沙门氏菌裂解抗原、鸭源鸡杆菌脂多糖抗原以及福氏志贺菌裂解抗原反应,仅与灭活的羊种布鲁菌(16M)发生反应。布鲁菌虎红平板凝集试验和试管凝集试验检测结果显示,获得的单抗可与标准检测抗原形成明显的颗粒凝集物和伞状凝集物。利用所建立的单克隆抗体细胞株,建立了一种检测布鲁菌的双夹心间接ELISA方法,并进行了特异性和敏感性检测。对模拟样品和临床样品进行检测,准确性均很高。     

小鹅瘟病毒VP3基因真核表达质粒在小鼠中的免疫效果

检测小鹅瘟病毒（GPV）VP3基因真核表达质粒在小鼠中的免疫效果。大量提取本实验室已构建的含有GPVVP3基因的真核表达质粒pVAXI／VP3和空载体pVAXI,分两组进行两点肌肉注射免疫小鼠,共免疫4次。利用MTY法检测免疫小鼠的T细胞增殖活性,间接ELISA法检测免疫小鼠血清中GPV特异性抗体的水平。淋巴细胞增殖指数,免疫小鼠与正常小鼠差异不显著。间接ELISA检测结果表明,pVAXI／VP3质粒免疫组小鼠血清中GPV特异性抗体水平,显著高于空载体对照组和阴性对照组。已构建的GPVVP3基因真核表达载体可以诱导小鼠产生明显的体液免疫,为小鹅瘟核酸疫苗的研究奠定基础。     

Preparation and evaluation of antigens used in serological tests for caprine syncytial retrovirus antibody in sheep and goat sera

Methods used to prepare antigens from caprine syncytial retrovirus (CSR) for use in the agarose gel immunodiffusion test (AGID) or an indirect enzyme-linked immunosorbent assay (ELISA) are described. Caprine and ovine sera were tested for antibody to CSR using the AGID test and ELISA incorporating a caprine system (CSR antigen and rabbit anti-goat IgG) or an ovine system (maedi-visna virus antigen and rabbit anti-sheep IgG). Good correlation was achieved in the results of the 3 tests when sera were devoid of antibody or were strongly positive. Variations in the results on weakly positive sera were considered to be more a matter of interpretation than due to basic differences in the reagents employed.     

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax)

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of ‘incomplete’ strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the ‘complete’ M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.     

Serological evidence of an Australian bovine lentivirus.

Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia.     

Detection of Dermatophilus congolensis antibody in the milk of streptothricosis infected cows

Passive haemagglutination and agar gel diffusion tests were used to detect specific antibody to Dermatophilus congolensis antigens in serum and milk of eight streptothricosis infected Friesian milking cows. All the sera and milk samples showed the presence of antibody but titres were higher in sera. Precipitating antibodies were detected only in three sera. A possible implication of this finding is discussed with respect to passive immunity in the young calves from infected dams.     

Immune complex-based vaccine for pig protection against parvovirus

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.     

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

Immune Complex‐based Vaccine for Pig Protection against Parvovirus

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK‐15 cell‐line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti‐PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC‐vaccines prepared in the form of a water‐in‐oil‐in‐water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long‐lasting anti‐PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two‐times higher content of the virus material administered by a commercially available vaccine. The IC‐based vaccines belong to non‐replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.     

Attempted definition by immunoblotting of the causes of reactivity in suspected false-positive sera in the Brucella ovis complement fixation test

Seventy-nine suspected false-positive sera, obtained over 1 year from routine submissions for Brucella ovis serological testing, were used in this study. These sera, which exhibited titres in the complement fixation test, but which because of their epidemiological history and their reactions in the enzyme-linked immunosorbent assay and gel diffusion test were suspected to be false positives, were further analysed by immunoblotting. In blots, using B. ovis antigens, rough lipopolysaccharide was identified as the major, immuno-reactive bacterial component. Antibodies against this macromolecule were present in 46.8% of the suspected false-positive sera. In order to find out if rough lipopolysaccharides from other bacterial species could be the possible cause for the suspected false positivity, 23 sera with highest complement fixation titres were reacted in blots with cell extracts from Escherichia coli, Yersinia enterocolitica, Yersinia pseudotuberculosis, Bortedella bronchiseptica, Actinobacillus seminis, Campylobacter fetus fetus, Campylobacter jejuni, Mycobacterium paratuberculosis, Mycobacterium phlei, Corynebacterium pseudotuberculosis and pure lipopolysaccharides from Escherichia coli and Salmonella typhimurium. Despite high frequencies of antibody reaction with proteins in most of these bacterial cell extracts, which reflect the presence of infections with these bacteria, immuno-staining in the rough lipopolysaccharide region was not observed.     

Serodiagnosis of Dermatophilus congolensis infection by counterimmunoelectrophoresis

Sixty-one sera from animals that had contact with Dermatophilus congolensis were examined by comparing three serological methods; counterimmunoelectrophoresis, passive haemagglutination, and agar gel diffusion, and by using four different antigenic extracts of D congolensis. The counterimmunoelectrophoresis was the most satisfactory of the methods having been found to be specific and sensitive, easy to perform and suitable for screening large numbers of samples. It was also found to have a higher antibody detection rate (82.2 per cent) than the other methods thus making it suitable for seroepidemiological surveys. It was found to be capable of detecting multiple antibodies and also revealed dissimilarities among the different antigenic extracts. The cellular antigens of D congolensis were found to detect antibody in more sera than the extracellular antigen; the cell wall extract proved to be the most satisfactory of all, detecting antibody from the largest number of sera compared to the other extracts in all the three serological tests.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.