Efficient generation of antibodies to oncoproteins by using synthetic peptide antigens.

To examine the efficiency of generating protein-reactive antipeptide antibodies, 35 peptides encoded by retroviral or cellular oncogenes were used to immunize rabbits. Thirty-two peptides elicited antipeptide antibodies, of which 56% reacted with their respective oncoproteins. The length of the immunizing peptide was an important factor in generating antibodies reactive with native protein. Similar peptides differing in a single or a few amino acids could elicite antisera of markedly different reactivities.     

Detection of anticentromere antibodies using recombinant human CENP-A protein

Objective. To evaluate CENP-A reactivity with anticentromere antibodies (ACA) using recombinant protein (rCENP-A). Methods. Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked immunosorbent assay (ELISA). Results. Of the ACA+ sera studied (n = 38), 95% were positive when tested against the rCENP-A in the ELISA system. Of the ACA- sera (n = 100), only 2% gave false-positive results in the assay. There was good correlation between the recombinant and bona fide antigens in assaying for ACA reactivity. Conclusion. CENP-A is a significant ACA target. The availability of the rCENP-A assay is a valuable adjunct to the previously described rCENP-B assay in analyses of the clinical significance of ACA.     

Specificity of anti-Sm antibodies by ELISA for systemic lupus erythematosus: increased sensitivity of detection using purified peptide antigens.

Sm antigen was purified by immunoaffinity chromatography using a murine monoclonal anti-Sm antibody and was confirmed to be free from contaminating polypeptides. This was then used to detect anti-Sm antibodies in patients' sera by enzyme linked immunosorbent assay (ELISA). Antibodies against Sm were detected in only 9/52 (17%) patients with systemic lupus erythematosus (SLE) by immunodiffusion, but 15/52 (29%) were positive for IgG anti-Sm antibodies by ELISA. The presence of anti-Sm antibodies remained disease specific despite the increase in sensitivity of this assay and validates its potential use for clinical application. There was no correlation between the presence of anti-Sm antibodies and any clinical features of SLE. In 23 renal biopsies a membranous component to the glomerulonephritis correlated with anti-Sm antibodies (p less than 0.05). Patients from West Africa, the Carribean Islands, and Asia had a higher prevalence of anti-Sm antibodies than the local Caucasian population.     

Detection and quantification of anti-ki antibodies by enzyme-linked immunosorbent assay using recombinant ki antigen

Objective. To establish an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Ki antibody, using a bovine recombinant Ki antigen, and studying its specificity. Methods. Sera from 220 patients with various connective tissue diseases were screened, and a prospective study of fluctuations in anti-Ki antibody and clinical course of a woman with systemic lupus erythematosus (SLE) was analyzed, by ELISA. Results. Anti-Ki antibodies were present in 18.9% of patients with SLE. The titer of anti-Ki antibody in the woman with SLE rose before the onset of pericarditis and pleuritis in this longitudinal study. Conclusion. ELISA using a recombinant Ki antigen is useful for the diagnosis of SLE, and it might be useful in estimating disease activity in patients with SLE.     

Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen.

OBJECTIVE. To establish an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Ki antibody, using a bovine recombinant Ki antigen, and studying its specificity. METHODS. Sera from 220 patients with various connective tissue diseases were screened, and a prospective study of fluctuations in anti-Ki antibody and clinical course of a woman with systemic lupus erythematosus (SLE) was analyzed, by ELISA. RESULTS. Anti-Ki antibodies were present in 18.9% of patients with SLE. The titer of anti-Ki antibody in the woman with SLE rose before the onset of pericarditis and pleuritis in this longitudinal study. CONCLUSION. ELISA using a recombinant Ki antigen is useful for the diagnosis of SLE, and it might be useful in estimating disease activity in patients with SLE.     

Analysis of human serum antibodies to human immunodeficiency virus (HIV) using recombinant ENV and GAG antigens

Recombinant proteins representing gag and env amino acid sequences of the Human Immunodeficiency Virus (HIV) (HTLV-IIIb) were produced in Escherichia coli and used to analyze sera for the presence of antibodies to HIV. ENV-9 is a protein representing the carboxy terminus of gp120 and part of gp41 which is highly immunoreactive. GAG-1 represents 83% and GAG-55 100% of the amino acids of the gag open reading frame. The purified proteins allow sensitive detection by enzyme linked immunosorbent assay (ELISA) of antibodies directed against either env or gag of HIV. We have determined the reactivity of sera from several HIV exposed individuals, either form high risk populations or with clinically defined conditions, in the ENV-9, GAG-55, and GAG-1 assays and found that two major seropositive groups are observed. The quantitative analysis of sera with env and gag antigens by ELISA showed AIDS patients had very low gag reactivity while retaining high env reactivity. Results obtained with authentic p24 viral protein in both ELISA and radioimmunoassay correlated to those from the GAG-55 ELISA. This correlation and the analysis of sera with both the ENV and GAG ELISAs indicate that the antibodies reactive to gag are specifically affected relative to env reactivity and that different levels of antibodies to separate viral components in these sera may correlate with disease state.     

人唾液、尿液中弓形虫抗原抗体的检测

目的探讨用唾液、尿液替代血清进行弓形虫感染免疫学诊断的可行性。方法用ELISA法对血清进行弓形虫循环抗原(CAg)、IgM、IgG抗体的检测,取上述各项免疫学指标阳性的血清样本80例,并随机抽取3项指标均为阴性的血清样本102例,分别与其唾液和尿液标本在同一反应板上同步进行测定,比较其阳性和阴性符合率。结果(1)554份血清样本中,CAg、IgM和IgG3项的阳性率分别为4.51%(25/554)、3.61%(20/554)、6.32%(35/554)。(2)唾液中检出IgM抗体阳性16例,与血清的阳性符合率为80%(16/20),检测CAg、IgG抗体均为阴性。(3)检测尿液中的抗原抗体均无一例阳性。结论采用唾液标本检测弓形虫IgM抗体,在弓形虫感染的诊断及流行病学调查中均具有一定的应用前景。     

Detection of P-glycoprotein in human leukemias using monoclonal antibodies

Summary Overexpression of a M_r 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines. Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein (>5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.     

A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera

Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.     

Detection of blood stage antigens of Plasmodium vivax by sandwich ELISA using pan-species monoclonal antibodies and polyclonal antibodies.

This paper reports an improved PcAb-McAb-ELISA test to detect blood stage Plasmodium vivax antigen in which the plates were coated with rabbit anti-P. cynomolgi polyclonal antibody to capture the antigens in test samples and two monoclonal antibodies, M26-32 and 3F9, were added together to react with the captured antigens. The coincidence rate with this test was 93% with microscopically confirmed P. vivax cases, 97% with normal samples, 95% with microscopically negative fever cases from nonendemic areas and 86% from endemic areas, respectively. The sensitivity was greater than 1 parasite/10(5) RBC.     

Isolation of antibodies specific to sickle hemoglobin by affinity chromatography using a synthetic peptide.

Antibodies to hemoglobin have been studied with a radioimmunoassay which employs [14C]carbamylated (= carbamolyated) hemoglobin S. An antiserum raised against hemoglobin S, which initially discriminated poorly between hemoglobins S and A, was fractionated by absorption to a column of Sepharose to which a synthetic peptide corresponding to the first 13 amino-acid residues of the beta chain of sickle hemoglobin had been covalently bound. A subpopulation of the antiserum was eluted from this column with 4 M guanidine - HC1. These antibodies showed binding to hemoglobin S but not to hemoglobin A and this interaction could be inhibited by the synthetic peptide. These antibodies, of demonstrated fine structural specificity, may be useful in the detection of sickle hemoglobin and in the study of its structure in solution.     

Detection of hepatitis C virus antibodies with new recombinant antigens: assessment in chronic liver diseases.

A new serological assay to detect antibodies against hepatitis C, based on a recombinant protein (BHC10) which incorporates structural and non-structural viral antigens, was tested in 67 healthy subjects and 409 patients with various forms of liver disease. Results were compared with the current assay based on the recombinant non-structural viral antigen c100 and with the recently introduced second-generation assay, Ortho2. None of the healthy subjects was positive by any of the assays. In patients with chronic non-A, non-B hepatitis the prevalence of anti-BHC10 was 96.8%, higher than anti-c100 (83.3%, p less than 0.001) and similar to Ortho2 (94.3%). False-positive results were less frequently found when BHC10 was used. These findings show that assays incorporating structural and non-structural antigens provide higher sensitivity to detect hepatitis C virus infection and they define an almost exclusive role of hepatitis C virus in the genesis of chronic non-A, non-B hepatitis.     

Serodiagnosis of tuberculosis: Detection of mycobacterial antibodies and antigens

Setting: The diagnosis of tuberculosis is based primarily on identification of mycobacteria and on clinical evidence. Recently, serological studies have been widely used experimentally as a diagnostic approach.Objective: The aim of our study was to optimize serodiagnosis of tuberculosis by detecting mycobacterial antigens and antibodies in sera from patients with lung tuberculosis, non-related diseases and healthy controls.Design: Mycobacterium tuberculosis H37Rv was disintegrated by pressure. Cell walls were extracted with 3 M KCL and were subjected to gel filtration in Toyopearl gel. Immune sera were prepared by immunization of rabbits with cell wall material. Anti H37Rv antibodies were purified by affinity chromatography. The reagents obtained were used to detect serum antibodies and antigens (following immune complex dissociation) using ELISA.Results: Using fraction 6 of cell wall extract, antibodies were detected in 72.2% of TB patients; there were no positive reactions in control subjects. By use of affinity-purified antibodies, antigens were detected in 77.1% of TB patients, 10% of patients with unrelated diseases and 6.7% of healthy controls.Conclusion: Effective serodiagnosis of tuberculosis can be achieved only by combining detection of both circulating antibodies and antigens using highly specific purified reagents and immune complex-dissociated sera.     

Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.     

Clinical significance of anti-Sm antibodies in systemic lupus erythematosus

Case records of 34 patients with systemic lupus erythematosus (SLE) were analyzed. Twelve patients had both anti-DNA and anti-Sm antibodies (Group I) and 22 had anti-DNA antibodies only (Group II). The disease patterns were comparable, except for (1) cutaneous vasculitis, which was observed in six of 12 patients in Group I and one of 22 in Group II (p less than 0.01); (2) pulmonary manifestations, nine of 12 in Group I and two of 22 in Group II (p less than 0.001); (3) cardiac manifestations, eight of 12 in Group I and four of 22 in Group II (p less than 0.01); and (4) renal biopsy, which showed milder lesions in Group I than in Group II (p less than 0.05). Evolution was fatal in four patients in Group I and in none in Group II. It is suggested that in SLE, the presence of anti-Sm antibody is associated with a much higher incidence of vasculitis, resulting in peculiar visceral manifestations, which can be poorly responsive to therapy. Whether there is a direct association between anti-Sm antibody and vasculitis or whether the common denominator is a genetic selection remains to be determined.     

Detection and quantification of thrombomodulin in human semen

The presence of fibrin degradation products, thrombin-like enzyme, prothrombin fragments, thrombin-activatable fibrinolysis inhibitor, plasmin and other active components of blood coagulation and fibrinolysis in seminal plasma has been reported. In the present study we investigate the presence of thrombomodulin in human semen. Using an Imubind thrombomodulin enzyme-linked immunosorbent assay (American Diagnostica Inc., Stamford, Connecticut, USA), seminal thrombomodulin levels were measured in 47 semen specimens obtained from subfertile individuals, normally fertile individuals, semen donors as well as vasectomized individuals, and in a further group defined by normality in several parameters derived from the World Health Organization fertility criteria. Conventional semen parameters were analysed in all semen samples. Thrombomodulin is quantifiable in human semen at a concentration lower than that normally found in citrated blood plasma samples. Slightly higher levels were seen for fertile stratifications compared with infertile individuals but without significant difference, given the numbers accrued. A vasectomized group showed the lowest value. In conclusion, our results establish the presence of thrombomodulin in human semen and suggest its production both upstream and downstream from the level of a vasectomy lesion.     

Detection of anti-Chlamydia trachomatis antibody by means of enzyme immunoassay using synthetic peptide antigen]

Newly developed diagnostic kits for the detection of Anti-Chlamydia trachomatis, Peptide-Chlamvdia (LOY: Meiji Milk Products Co., Ltd., Tokyo; for IgG and IgA), were evaluated using the microimmunofluorescence assay (MIF) as the gold standard. These results were also compared to results of testing by Sero-IPALISA and immunoblot (I-B). Detection by LOY in based on enzyme immunoassay with synthetic peptides as the antigen. Thirty serum samples from pediatric patients and 130 serum samples from gynecology patients were used. All 26 pediatric samples that were positive for Chlamydia pneumoniae IgG antibody tested negative with LOY, indicating that the presence of the antibody against C. pneumoniae did not affect the assay by LOY. For 90 gynecological samples, the total, the positive and the negative agreement rates for IgG were quite high; i.e. 87.8%, 90.0% and 70.0% (LOY vs MIF), 85.6%, 85.0% and 90.0% (Sero-IPALISA vs MIF), and 92.0%, 94.9% and 70.0% (I-B vs MIF), respectively. On the other hand, many cases of MIF (-) and LOY (+) discrepancy were seen in IgA detection. In order to better understand the basis for such disagreement. 34 serum samples were collected from patients whose cervical samples were negative for the Chlamydia group antigen based on the assay with IDEIA-Chlamydia. They were then assayed by MIF and LOY. The total, the positive and the negative agreement rates for IgG were 91.2%, 100% and 90.9%, while the total and the negative agreement rates for IgA were 88.2% and 88.2% (there were no IgA positive cases). Furthermore, 6 serum samples (1 case of MIF (+) LOY (+) and 5 cases of MIF (-) LOY (+)) were provided to determine whether LOY detects C. trachomatis specific IgA antibody. Increasing amounts of C. trachomatis serovar L2 were added to the serum samples resulting in a progressive decrease in their reactivity in the LOY assay. These results lead us to speculate that LOY can reveal even low levels of C. trachomatis specific IgA antibody. In conclusion, LOY can be used as an useful kit for detecting C. trachomatis antibody.