Two-site pan-species monoclonal antibody ELISA for detection of blood stage malaria antigen.

A two-site pan-species monoclonal antibody sandwich ELISA (MAb-MAb ELISA) was developed to detect both Plasmodium vivax and P. falciparum antigens in whole blood impregnated on filter paper. In this assay, the plates were coated with pan-species MAb 3F9 and another pan-species MAb M26-32 conjugated with alkaline phosphatase was used for detection of bound antigen. The sensitivity of this assay was 5, 10 and 10 parasites per 10(6) erythrocytes for cultured P. falciparum, patient-derived P. vivax and P. falciparum, respectively. The coincidence rates for this assay were 93% (92/99) with healthy individuals and 93% (42/45) with microscopically confirmed vivax malaria cases. After two weeks treatment, 77.7% (14/18) of vivax malaria were still positive by this assay but with diminished level of reactivities [corrected].     

Sandwich ELISA detection of excretory-secretory antigens of Toxocara canis larvae using a specific monoclonal antibody

We produced a new monoclonal antibody (mAb) to the excretory-secretory (ES) antigens of Toxocara canis larvae. The mAb (IgG1) reacts specifically with the 120 kDa protein of many ES molecules and does not have any cross-reactivity with adult T. canis antigens. Sandwich ELISA to detect the ES antigens was performed using the mAb and rabbit polyclonal antiserum. The lower limit for the detection of ES antigen was 4 ng/ml; assay was proportional within a concentration range of 4 ng/ml to 1 microg/ml of ES antigen. This assay system may prove valuable when seeking to quantify parasite burden early in infection and when determining the efficacy of anthelmintic treatment.     

抗ALT抗体间接ELISA检测方法的建立

目的建立免疫小鼠体内抗人丙氨酸氨基转移酶(ALT)抗体的间接ELISA检测方法,以期作为融合后杂交瘤细胞株的筛选体系。方法利用人ALT为包被抗原,以1∶40 000稀释的辣根过氧化物酶标记的羊抗鼠IgG为二抗,采用棋盘滴定法确定抗原和对照血清(阴性、阳性)最佳工作浓度及ELISA封闭液的浓度建立ELISA体系。结果包被抗原和血清的适宜浓度或稀释度分别为1∶5 000和1∶1 000;用4%BSA封闭18 h~24 h效果最好。结论建立的ELISA方法稳定,简便易行,能准确检测免疫小鼠血清抗体的效价,并可用于抗ALT单克隆抗体杂交瘤细胞株的筛选。     

Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

AIM:To establish an ELISA kit using monoclonal antibodiesagainst Clostridium difficile ( C.difficile) toxin A.METHODS:An indirect sandwich ELISA was described usingthe purified rabbit monospecific antiserum as capturingantibody.After the polystyrene microtitre plates with 96flat-bottomed wells were coated with rabbit antiserum,the wells were blocked with 100 g/L BSA in PBS-T.C.difficiletoxin A or culture filtrates were added to each well and thenmonoclonal antibodies IgG-horseradish peroxidase conjugatewas added as detecting antibody,tetramethylbenzidine wasused as substrate and A_(450) of the stopped reacting productwas recorded in an automated plate reader.RESULTS:The tested specimens included culture filtratesof 2 strains of toxigenic C.difficile,2 strains of non-toxigenicC.difficile,26 strains of E..coli,2 strains of S.dysenteriae,1 strain of Bif.infantis,5 strains of V.cholera,2 strains ofS.typhi,7 strains of C.botulinum,1 strain of toxigenicC.sordllii,and 1 strain of C.butyricum.A total of 47 strainsof culture filtrates were all negative except for 2 strains oftoxigenic C.difficile.The detective limitation of toxin A was0.1 ng/mL.CONCLUSION:An ELISA kit with high specificity andexcellent sensitivity for the rapid detection of C.difficiletoxin A was established.It will be a useful tool for diagnostictest of C.difficile toxin A.     

Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C. difficile) toxin A. METHODS: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL. CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.     

抗疟原虫单抗 M26-32IgM 的单体化及其免疫学特性研究

目的改造IgM类抗疟原虫单抗M26-32为单体IgM亚单位，胶体金标记天然和改造后两种M26—32用于检测疟原虫抗原。方法复苏培养M26—32杂交瘤细胞，接种BALB／c小鼠，收集含有单抗的腹水，用商品HiTrap IgM HP柱纯化腹水中的IgM抗体，并用L-半胱氨酸裂解IgM类M26—32单抗成单体IgM亚单位。经间接免疫荧光检定两种抗体的效价，并用胶体金标记，分别检测恶性疟原虫和间日疟原虫可溶性抗原。结果接种20只小鼠，共采集约80mL单抗腹水，经HiTrap IgM HP柱纯化，获得了纯度较高的M26—32单抗。经L一半胱氨酸裂解后，Native电泳显示天然IgM被裂解为单体亚单位，IFA结果显示，亚单位M26-32与天然五聚体IgM滴度无差别。经胶体金标记后，两种金标抗体均分别能检测1：100稀释的恶性疟原虫和1：10稀释的间日疟原虫可溶性抗原。结论建立了L-半胱氨酸裂解IgM类M26—32单抗为单体IgM亚单位及其胶体金标记的方法，为利用M26—32进一步制备相应快速诊断工具奠定基础。     

The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission.

The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P. falciparum glutamate-rich protein, GLURP489-1271, expressed as an E. coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission. Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing the antigens, the responses were often short-lived. In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens. The antibody response, even against large fragments of conserved antigens, is not uniformly elicited by natural malaria infection in previously primed donors.     

Evaluation of the efficiency of rapid tests to detect malaria patients and parasite carriers in Tajikistan

The sensitivity of a rapid test versus microscopy of blood samples was studied while examining 7983 dwellers from the republic's endemic areas. The results of blood testing in the Pyanj and Shurobod districts showed agreement with those of blood microscopy. However, its sensitivity in two other districts was as high as 1.9%. Only one of 53 positive tests was verified by microscopy examination. The findings suggest that it is impossible to use the examined tests (Access Bio Inc. Care Start Malaria pLDH 2 line test) under the conditions of Tajikistan; all the more they identify malaria without differentiation in its pathogen types.     

Field evaluation of an ELISA to detect antibody in leprosy patients and their contacts

Previous studies have detected circulating antibody in leprosy using a variety of difficult laboratory methods. We have developed a simpler method for detecting antibody by ELISA, using autoclaved Mycobacterium smegmatis as the antigen. Evaluation was performed on eluates from 25 microliter aliquots of finger-prick blood dried on filter-paper disks in two high-incidence populations in Ponape, Micronesia. Among 228 nonleprosy cases bled in 1980 and rebled and re-examined in 1982: a) for those who had been ELISA positive two years earlier, the leprosy attack rate during the intervening two years was at least twice as high as among those who had been negative, and we estimate that shortening the screening interval to one year plus doing confirmatory retests on new sero-converters would increase the relative risk (or "predictive power") to over sixfold, including all impending multibacillary cases; b) elevated antibody levels were detected up to two years prior to clinical onset of disease in 70% of new cases; and c) both asymptomatic conversion (rising titer) and reversion (falling titer) were observed. Among 150 biopsy-proven cases, ELISA results suggest that fall of titer in most uncomplicated paucibacillary cases was rapid (months), but in multibacillary cases was more gradual (years), probably paralleling responses to treatment with titers rising in reactivation. These results suggest that this technique, with an improved antigen, may be useful in leprosy control programs, both for detecting candidates for preventive treatment and for following responses to therapy.     

HLA antigens and adult rheumatoid arthritis: a study with a monoclonal antibody

Adult rheumatoid arthritis (RA) is a very heterogeneous disease that is associated with HLA-antigens, although no absolute association has been found with any particular HLA type. Forty-one seropositive RA patients have been studied with a local monoclonal antibody named X1 21.4 (9w940), strongly associated with HLA-DRI, DR4, Drw10 antigens, to verify a possible correlation with the disease. The results obtained have also been compared with the data reported on MC1, a serologically defined determinant correlated with RA. X1 21.4 monoclonal antibody appears to be associated with the disease and it could identify one epitope involved in the susceptibility to RA.     

Detection of P. vivax antigens in malaria endemic populations of Nepal by ELISA using monoclonal antibodies raised against Thai isolates

An indirect enzyme linked immunosorbent assay (ELISA) using monoclonal antibody (MAb) originated from the native Thai isolates of P. vivax (McPV1) and the polyclonal antibody (PAb) raised against Nepali isolates of P. vivax was developed for detection of P vivax antigens in red cell lysates. The assay was specific (100%) since it was positive only with P. vivax-infected erythrocytes and was negative when erythrocytes from 40 healthy individuals from malaria non-endemic areas and 40 P. falciparum infected erythrocytes were tested. When the assay was applied to 203 vivax blood samples already proven by microscopic examination collected from Dhanusha district of Nepal, and using the cut-off level of the mean optical density (OD) (0.144) of 40 healthy individuals who had been living in malaria-endemic areas (0.073) + 2 SD (0.016), the assay could detect 189/203 samples, indicating the sensitivity of the test was 93.1% with a detection limit of erythrocytes of 240 parasites/10(6) erythrocytes. In addition, the assay was negative when 40 blood samples with fever of unknown origin, collected from the same malaria-endemic areas, were tested. However, there was a significant correlation between OD values and parasitemia (r=0.649; p=0.018). The results indicate that MAb-PAb indirect ELISA using MAb raised against Thai isolates of P. vivax as the coating antibodies, and polyclonal antibodies raised against local Nepali isolates as the detecting antibody, could detect P. vivax antigens with high degrees of sensitivity and specificity. Furthermore, it seems that the McPV1 MAb raised against Thai isolates of P. vivax could recognize the antigens of Nepali isolates in a wide range of blood samples.     

In-situ immuno-PCR to detect antigens

In-situ immunoassays do not allow the detection of the minute numbers of target molecules accessible with in-situ PCR. We developed a highly sensitive method, termed in-situ immuno-PCR, in which the DNA marker was linked to target molecules through an antibody-biotin-avidin bridge and amplified by in-situ PCR. Amplified DNA sequences were detected in situ by hybridisation. This technique may be the only one available to detect minute quantities of biological macromolecules such as proteins, carbohydrates, and lipids in intact cells or tissue sections.     

A monoclonal antibody to hemoglobin F

Summary A monoclonal antibody BMU7-17 which recognizes an antigenic site unique to the cyanogen bromide fragment, CB2 (residues 56 through 133) of the -chain of human hemoglobin has been produced using cell hybridization techniques. The antibody does not crossreact with hemoglobin A, the isolated - or -chains, or with hemoglobin of various mammals. Affinity chromatography on Staphylococcal protein A-Sepharose 4B and radial immuno-diffusion revealed the antibody to be of the mouse IgG 1 class. BMU7-17 identifies F cells in adult and in cord blood specimens, as demonstrated by an indirect immunofluorescence assay. The dissociation constant of the antibody as determined using tritiated hemoglobin F with a double antibody radioimmunoassay has a K_d=1.2×10^–9 moles by Scatchard plot analysis.     

A monoclonal antibody to surface antigens on microfilariae of Brugia malayi reduces microfilaremia in infected jirds

A monoclonal antibody to surface antigens of Brugia malayi microfilariae promotes the adherence of peripheral blood cells to microfilariae in vitro and reduces microfilaremia in vivo. The antibody reacts with epitopes present on two stage-specific antigens with estimated molecular weights of 70,000 and 75,000 daltons.     

An immunoassay to detect human embryonic epsilon globin chains by a murine monoclonal antibody

Human embryonic epsilon globin chains were isolated from lysates of cultured human leukemic K562 cells induced with hemin. Mice were immunized with epsilon globin chains, and a murine hybridoma cell line was established that secreted monoclonal anti-epsilon globin antibody. A sensitive and specific slot blot immunoassay for epsilon globin chains has been developed. epsilon Globin chains were shown to be present in hemolysates from fetuses of 20 weeks' gestation but were not detected in normal cord blood hemolysates. This immunoassay is useful for the study of epsilon globin ontogeny during embryonic and fetal development as well as for searching for inappropriate epsilon globin chain expression in hematologic disorders resulting from genetic abnormalities or neoplastic processes.