Enzyme immunoassay of the level of parasite-specific IgE antibodies in schistosomiasis using the monoclonal antibody BL-IgE 9

Sera from patients with chronic schistosomiasis (Schistosoma mansoni or Schistosoma haematobium) were examined for the presence of parasite specific IgE antibodies by means of ELISA technique using tegument antigen prepared from adult worms of Schistosoma mansoni and using the monoclonal antibody BL-IgE 9. Individuals from tropical countries who had no schistosomiasis and blood donors from GDR were studied for comparison. Significantly higher levels of specific IgE antibody were given by sera from patients with schistosomiasis than by the controls. These differential responses serologically differentiated between patients with chronic schistosome infections and noninfected individuals.     

Clinical features and parasite-specific IgM/IgG antibodies of paragonimiasis patients recently found in Japan

Clinical features of a total of 30 paragonimiasis westermani patients referred to and diagnosed in our laboratory in 1999 were analyzed retrospectively. Most patients were middle-aged (average: 48 years, range: 13-72 years) with the male/female ratio of 19/11. Over 70% of the patients had respiratory symptom and over 80% had peripheral blood eosinophilia and high serum IgE level. All but two cases had radiologic abnormalities on the chest X-ray. Only in 3 cases were Paragonimus eggs detected in the sputum smear. We classified the patients into two groups depending on the chest X-ray findings: patients having pleurisy alone and those having nodular/cavitating lesions in the lung parenchyma. We measured parasite specific IgM/IgG antibodies in all patients sera by microplate ELISA. The mean parasite-specific IgM/IgG antibody ratio was significantly higher in the parenchymatous lesion group than in the pleurisy group. While IgM antibody titer had a strong positive correlation with the degree of eosinophilia in peripheral blood, IgG antibody titer had an inverse correlation. Although the degree of eosinophilia in peripheral blood was higher in the pleurisy group than in the parenchymatous lesion group, total IgE level in serum was comparable between the two groups. The present results indicate that pleurisy with eosinophilia and dominant IgM antibody are the characteristic features of the early stage of paragonimiasis, whereas parenchymatous lesions in lungs with low grade eosinophilia and dominant IgG antibody are of the late stage. These results suggest that detection of IgM antibody should always be considered for the immunodiagnosis for paragonimiasis-suspected patients with pleurisy.     

Biological activity of human-mouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity.

Chimeric antibodies were constructed in which the murine variable region of anti-colorectal cancer monoclonal antibody CO17-1A was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions. Human-mouse chimeric proteins were compared with the parental murine IgG2a antibody CO17-1A for their ability to participate in tumor-cell destruction by human and murine effector cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. All of the chimeric antibodies showed different degrees of ADCC with human lymphocytes, monocytes, and granulocytes and with murine macrophages. Monocytes and macrophages were able to utilize the chimeric IgG1 and, to a lesser degree, IgG4 and IgG3 antibodies to lyse tumor-cell targets in ADCC assays. The chimeric IgG1 and IgG4 antibodies were nearly as effective as the parental CO17-1A antibody in inhibiting tumor growth in nude mice. These data indicate that chimeric IgG1 antibody is superior in its antitumor activity.     

Detection of a circulating antigen in human schistosomiasis japonica using a monoclonal antibody

A double antibody enzyme-linked immunosorbent assay (sandwich ELISA) was used for the detection of a circulating antigen from human schistosomiasis japonica infections. This assay involves the use of polyclonal rabbit Schistosoma japonicum soluble egg antigen (SEA) antiserum to bind circulating antigen and a monoclonal antibody (MabH4) to identify and quantify this antigen. Sera from 108 S. japonicum-infected patients (acute and chronic) were tested. Sera from 93 of 95 patients with chronic infection were positive for this antigen; sera from 12 of 13 patients with acute infections were also positive. Antigen was not detectable in control human sera. Sera from 35 chronic schistosomiasis patients were collected 6-12 months after praziquantel treatment. Circulating antigen was not detectable in the sera of 33 of these patients and was dramatically reduced in 2. This ELISA system may prove valuable in differentiating past and current infections.     

Detection of circulating antigen in the sera in patients with schistosomiasis using monoclonal antibody testing

Monoclonal Antibody-Antigen Spot Test (McAb-AST) was used for detecting circulating antigen in sera from patients with schistosomiasis japonicum. McAb-AST positive rate in 308 schistosomiasis patients before treatment was 75.97%. Among the patients who had moderate or severe infection, the McAb-AST positive rates were 91.18% or 100% respectively. However, the McAb-AST cross-reactive rates in 123 normal donors, 98 clonorchiasis patients, 50 paragonimiasis patients and 56 patients with other parasitic diseases were 0%, 3.06%, 4%, and 1.79% respectively. 1.3 and 6 months after treatment, the McAb-AST negative-converting rates in patients who had negative results of stool hatching were 98.03%, 100% and 100% respectively. Compared with the results of detection of circulating antibodies, negative-conversion of circulating antigen in the patients with negative stool hatching appeared earlier and the negative-converting rate of circulating antigen was much higher. Therefore, McAb-AST had significant value in clinical practice because of its high sensitivity, specificity and good reproducibility.     

Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies

Two murine monoclonal antibodies, FMC 25 and AN 51, directed against distinct epitopes on the glycoprotein Ib complex, have been used to further define the mechanism of quinine/quinidine drug-dependent antibody interaction with platelets. FMC 25, directed against an epitope on glycoprotein IX, had no effect on platelet aggregation induced by collagen or adenosine diphosphate and little, if any, effect on ristocetin-induced platelet agglutination. FMC 25 and its (Fab)2 fragment, however, were potent inhibitors of drug-dependent antibody- induced platelet aggregation and blocked binding of drug-dependent antibody to platelets as assessed by indirect platelet immunofluorescence. In contrast, AN 51, directed against an epitope on the alpha-subunit of glycoprotein Ib, blocked ristocetin-induced, factor VIII/von Willebrand factor (FVIII/vWF)-dependent platelet agglutination but not drug-dependent antibody-induced platelet aggregation or binding of drug-dependent antibody to platelets. Selective proteolytic removal of the majority of the alpha-subunit of glycoprotein Ib (glycocalicin) from platelets by treatment with calcium- dependent protease did not affect binding of drug-dependent antibody. In addition, a quinidine-dependent antiplatelet antibody immunoprecipitated glycoprotein Ib complex from normal platelets and the membrane-associated proteolytic remnant of the glycoprotein Ib complex from calcium-dependent protease-treated platelets. Preincubation of drug-dependent antibody with purified glycoprotein Ib complex inhibited subsequent binding of antibody to platelets, but the separated components, glycoprotein Ib and glycoprotein IX, were both ineffective, suggesting that the normal interaction between glycoprotein Ib and glycoprotein IX in the intact complex was necessary for drug-dependent antibody recognition. The functional response of platelets to drug-dependent antibody was not mediated by way of platelet Fc receptor, since aggregation of washed platelets by acetone- aggregated IgG was not inhibited by FMC 25 (Fab)2. FVIII/vWF was not required for drug-dependent antibody-induced platelet aggregation. The combined evidence is consistent with quinine/quinidine-dependent antibody-platelet interaction occurring by way of a FVIII/vWF- independent, Fc receptor-independent mechanism that probably involves binding of antibody to glycoprotein IX or the beta-subunit of glycoprotein Ib or both.     

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).     

Immunological activities of monoclonal IgG1 antibody against Trypanosoma gambiense.

The present paper deals with the immune reaction between a monoclonal IgG1 antibody and Trypanosoma gambiense. The aggregation of trypanosomes, immune adherence to macrophages and protection against infection are associated with the antibody. IgG1-mediated clumping of trypanosomes is readily dissociated by the addition of complement. Dissociation of the clumped trypanosomes in the equivalence area released approximately fifty percent of previously bound surface antigens. These antigens were capable of binding again to new IgG1 antibody. Complement deposition rendered bivalent IgG1 antibody in the immune complex functionally univalent. Such an event in the presence of complement is of great advantage to the infected host in killing pathogens in vivo, as it allows more antibodies to attach to surface antigens and subsequently to initiate complement activity.     

Crossreaction of monoclonal antiidiotypic antibodies specific for human antithyroglobulin antibody with the Fc portion of human IgG

We established 2 monoclonal antiidiotypic antibodies that reacted not only with a private idiotope on human antithyroglobulin antibody but also with the Fc portion of human IgG. These antiidiotypic antibodies (B2 and B6) bound to IgG F(ab')2 antithyroglobulin derived from a patient with chronic thyroiditis and normal human IgG, but did not bind to IgG F(ab')2 nonantithyroglobulin derived from the patient and normal human IgG F(ab')2. The antiidiotypic properties and the rheumatoid factor (RF) like properties of B2 and B6 were confirmed by inhibition assay. Our results suggest that RF may be produced as antiidiotypic antibodies specific for other antigens than IgG.     

Enzyme-linked immunosorbent assay using monoclonal antibodies for direct serotyping of enteric adenoviruses in feces

We were prepared three monoclonal antibodies in which the monoclone 12D was type specific for Adenovirus 40 (Ad40), 1F was type specific for Ad41 and 15D was group specific for Ads. For identification of enteric adenoviruses (EAd) in stool specimens, enzyme-linked immunosorbent assay (ELISA) test using monoclonal antibodies was developed. Results of identification by the ELISA tests using monoclonal antibodies to EAd on 15 fecal samples in which Ad particles were found by electron microscopy showed complete coincidence to those of Sma 1 restriction endonuclease cleavage. From these results, the ELISA tests employing EAds type specific monoclonal antibodies proved to be specific and this was a rapid technique for laboratory diagnosis of EAd in fecal specimens of viral gastroenteritis. Fifty-eight fecal samples with Ad particles positive by EM were serotyped by the ELISA using monoclonal antibodies. Eleven fecal samples were identified as Ad40, 25 as Ad41, 1 as double infection with Ad40 and Ad41, and 4 as non-EAd. These results indicated that Ad41 was more dominant than Ad40 during April, 1986 to January, 1989 in Matsuyama city.     

Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.     

A two-color flow cytometry assay for detection of hairy cells using monoclonal antibodies

We have developed a simple two-color immunofluorescence assay equally suited for microscopy and flow cytometry detecting hairy cells (HCs) in single cell suspensions, based on the concomitant reactivities with the B cell-specific monoclonal antibody B1 (CD20) and the monocyte/HC- associated antibody SHCL-3 (CD11c). Thus, HCs can be demonstrated in peripheral blood, bone marrow, and spleen specimens from hairy cell leukemia (HCL) patients even when they constitute less than 1% of the cell suspension. Likewise, admixture experiments with normal mononuclear cells and the MOLT-4 T-acute lymphocytic leukemia (ALL) cell line demonstrated that HCs could be detected in amounts as low as 1%. The validity of this assay has been ascertained by the lack of double marker positivity in cell suspensions from B-chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) patients that only expressed B1 or SHCL-3, respectively. Furthermore, other malignant blood diseases, including malignant lymphomas, acute leukemias, and chronic leukemias disclosed no double marker positive cells. In a clinical setting, this assay was used for purifying HCs (by flow cytometry) from the peripheral blood from patients with no apparent morphological evidence of circulating HC infiltration and for monitoring the effect of interferon therapy. In conclusion, this assay should be of value for both diagnosis and monitoring patients with HCL.     

An enzyme-linked immunosorbent assay for the measurement of thyroglobulin in human serum using mouse monoclonal antibodies

A robust, rapid, sensitive and specific enzyme-linked immunosorbent assay (ELISA), using an in-house immunoglobulin-A-sub-class mouse monoclonal human-thyroglobulin antibody (WNSM2), with a sensitivity of 1.51 pmol/l has been established for the measurement of thyroglobulin in serum. Standard curves in varying dilutions of human serum were similar to standard curves obtained in serum-free medium, thus demonstrating no significant cross-reactivity with any serum proteins other than thyroglobulin. Levels of serum thyroglobulin detected by ELISA correlated significantly (r = 0.93, P less than 0.001) with those from a standardized and well-characterized radioimmunoassay. The coefficients of variation within and between ELISA assays were 3.9 and 7.1% respectively. Thyroglobulin was detectable in 87% of 54 normal subjects who had no history of thyroid or autoimmune disease, the mean (+/- S.D.) for this group being 15 +/- 6.6 pmol/l with a range of 1.51-53 pmol/l. Using this assay, levels of thyroglobulin were shown to be significantly (P less than 0.002) increased in patients with untreated hyperthyroid Graves' disease compared with normal subjects.     

A two-colour immunofluorescence test with a monoclonal human proinsulin antibody improves the assay for islet cell antibodies

Summary The conventional indirect immunofluorescence assay for islet cell antibodies was compared with a two-colour immunofluorescent assay to detect both islet cell antibodies with fluorescein isothiocyanate-labeled rabbit anti-human IgG and pancreatic B cells with a monoclonal human proinsulin antibody and Texas red-labeled sheep anti-mouse IgG. Determinations of end-point titres showed a correlation between the new two-colour immunofluorescent assay and the conventional indirect immunofluorescent assay in 1) selected sera positive for islet cell antibodies and insulin autoantibodies r _s= 0.93 (p<0.01) or for islet cell antibodies alone r _s=0.99 (p<0.005) and 2) sera from children or young adults with newly diagnosed Type 1 (insulin-dependent) diabetes r _s=0.95 (p<0.0001). No interference between the monoclonal human proinsulin antibodies and islet cell antibodies with or without insulin autoantibodies or between the two second fluorescent antibodies was detected. It is concluded that the two-colour immunofluorescence assay is advantageous since a) it is possible to mix the reagents to avoid a more time-consuming and technically complicated assay, b) the presence of B cells can be confirmed in each section to permit detection of B cell cytoplasmic antibodies and c) microscopic evaluation is easier and more accurate, particulary in islet cell antibody negative samples.     

Development of immunoradiometric assay for detection of Plasmodium falciparum antigen in blood using monoclonal antibody

Specific monoclonal antibody (MAB) F2W22C1) produced by hybridoma technology against blood stage common antigens shared by almost all isolates of P. falciparum was selected. The 125I-labelled prepared from this MAB (125I-MIgG) was used as probe for detection of low grade P. falciparum infection. Recently, a two-site monoclonal antibody sandwich immunoradiometric assay (Mab-IRMA) has been developed. The assay showed good correlation with parasitaemia with the ability to detect as few as 2.4 parasites/10(8) erythrocytes. Two surveys were made in people living in a malaria endemic area during transmission and non-transmission season to detect the antigen of Plasmodium falciparum by using the Mab-IRMA. In the first survey involving 101 people, the IRMA positive rate was 56.4% and then significantly declined to 16.5% during the second survey involving 79 people of the same group. The parasitological positive rates were likewise decreased from 11.9% to 1.3% (p = 0.015) during these two seasons. IRMA positive rates were significantly higher than the corresponding parasitological positive rates (p less than 0.0001 and less than 0.002 for the first and the second surveys respectively). Regression analysis showed that IRMA activities were linearly correlated with the parasite counts by microscopic examinations (r = 0.629, p = 0.022). IRMA was specific for P. falciparum since all 30 healthy controls and six of seven vivax malaria cases were negative. IRMA has potential for use to monitor the malaria control and to assess the efficacy of the future field trial of malaria vaccine.     

Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.