Anti-skeletal muscle and anti-acetylcholine receptor antibodies in patients with thymoma without myasthenia gravis: relation to the onset of myasthenia gravis.

We measured the anti-skeletal muscle (SM) antibody titers in sera from 46 patients with thymoma but without Myasthenia gravis (MG) in order to determine whether the presence of anti-SM antibody is linked to the combination of thymoma-MG, or to thymoma alone. We detected anti-SM antibodies in 18 of these sera, of which 15 had concomitantly elevated titers of anti-AChR antibodies. Moreover, 9 of whom had experienced the onset of MG after surgery. In contrast, no patient without elevations in both antibodies developed MG during the followup. We conclude that the presence of anti-SM antibodies is linked strongly with thymoma associated with MG, but not with thymoma alone.     

Free PSA forms in prostatic tissue and sera of prostate cancer patients: analysis by 2-DE and western blotting of immunopurified samples

OBJECTIVE: The isoform pattern of free prostate-specific antigen (fPSA) from sera of patients with prostate cancer (PCa) and no evidence of prostate cancer (NPCa), cancerous and non-cancerous tissues and seminal plasma, have been compared, above all regarding the degree of sialylation, with the aim to show a better discrimination of PCa and NPCa. DESIGN AND METHODS: The samples have been immunopurified and analyzed by two dimensional gel electrophoresis and western blotting. It was investigated which patterns were obtained when looking for the fPSA and the (-5/-7)proPSA (precursor form) before and after desialylation. RESULTS: The fPSA sialylation and the proPSA pattern in cancerous and non-cancerous prostate tissues were similar to each other and only slightly different from PCa and NPCa sera. The different fPSA isoforms observed could not be due solely to differences in the degree of sialylation, because different fPSA and (-5/-7)proPSA precursor isoforms were still present after complete desialylation. CONCLUSIONS: Although slight differences in the fPSA and (-5/-7) proPSA glycosylation and isoform pattern were observed, they were not large enough to be considered to improve PCa diagnosis.     

Myasthenia gravis, muscle twitch, hyperhidrosis and limb pain associated with thymoma: proposal of possible new myasthenic syndrome.

We describe a 54-year-old man with myasthenia gravis, thymoma, systemic muscle twitch particularly of both lower limbs, hyperhidrosis and lower limb pain. The muscle twitch resembled to fasciculation rather than to myokymia and was persistent after discontinuation of anti-acetylcholinesterase drug. No attenuation nor disappearance of the muscle twitch was educed by spinal anesthesia. However, it disappeared when a nondepolarizing type muscle relaxant (pancuronium bromide) was used. The muscle twitch was thus considered to originate from peripheral axons. Thymoma was considered to be involved in the pathogenesis of these unusual clinical manifestations which may constitute a new myasthenic syndrome.     

Anti-brush border antibodies (ABBA) in sera from patients with ulcerative proctocolitis and in sera with antibodies against Yersinia enterocolitica 0:3

20 serum samples from patients with ulcerative proctocolitis (UC), and 20 serum samples with previously demonstrated IgG antibodies against Yersinia enterocolitica serotype 0:3 were analyzed with respect to the occurrence of anti-brush border antibodies (ABBA). 50% of the UC-sera and 20% of the anti-0:3 sera were ABBA-positive. Occurrence of ABBA did not apparently correlate to disease activity, or disease extension, or medication.     

Detection of varicella-zoster virus-specific IgA antibodies in varicella and zoster patients and in healthy adults of various ages by solid-phase radioimmunoassay

The varicella-zoster virus(VZV)-specific serum immunoglobulin A(IgA) response was studied in varicella and zoster patients and in healthy adults of various ages by a sensitive solid-phase radioimmunoassay technique. Significant rises in VZV-specific IgA titer were found for both varicella and zoster patients, while VZV-specific IgA was also detected in greater than 90% of healthy adults but at lower titers. No significant decrease in VZV-specific IgA titer with increasing age was found. The role of humoral versus cell-mediated immunity in VZV reactivation is discussed.     

Assays for thyrotropin-receptor binding and thyroid-stimulating antibodies in sera from patients with Graves' disease

We compared the activities of thyroid-stimulating antibodies (TSAb) measured in cultures of human thyrocytes and the values for thyrotropin-receptor antibodies (TRAb) as measured with a commercial kit based on use of radiolabeled receptors. Sera were obtained from patients with Graves' disease before, during, and after therapy with carbimazole (1-methyl-2-thio-3-carbethoxyimidazole). We found a significant correlation between the measurements of these two antibodies in patients: before treatment (r = 0.74, p less than 0.01, n = 44), after three months of treatment (r = 0.76, p less than 0.01, n = 21), and during relapse after the drug was discontinued (r = 0.64, p less than 0.01, n = 19). In all three situations, our TSAb technique was more sensitive than the TRAb method. We conclude that, even though the TRAb technique is simpler and quicker, this commercial kit is too insensitive to replace measurement of TSAb in fresh human thyrocyte cultures for management of drug therapy of patients with Graves' disease.     

Monoclonal antibodies reacting with placental protein 5: use in radioimmunoassay, Western blot analysis, and immunohistochemistry

Monoclonal antibodies were raised reacting with placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Immunization was carried out with an antigen purified from late pregnancy placenta tissues. After fusion with myeloma cells, clones producing antibodies reacting with PP5 were isolated. Antibodies produced by two of the established hybridoma clones were characterized. The Ka of the antibodies was 0.22 x 10(9) L/mol and 0.3 x 10(8) L/mol. in Western blot analysis, both monoclonal antibodies reacted with the purified antigen that had a relative molecular weight (Mr) of 30 kd, but minor components of Mr 27 kd, 56 kd, and 62 kd were also identified. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, the purified protein yielded three polypeptides (Mrs of 16.4 kd, 16.8 kd, and 18.3 kd) that did not react with the monoclonal antibodies in Western blot analysis. By immunoperoxidase staining with monoclonal and polyclonal antibodies, PP5 was localized to the syncytiotrophoblast, cytotrophoblast, and endothelium of early and late pregnancy placenta tissues, whereas various other tissues were PP5-negative. In immunofluorescence staining, isolated endothelial cells were stained with both monoclonal antibodies. Endothelial cells in monolayer culture released into the medium a substance that is immunologically similar to purified PP5.     

Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane

Previous studies have demonstrated that antibodies in sera from patients with halothane hepatitis recognize halothane-induced liver microsomal polypeptide neoantigens, and have suggested that these antibodies may play a role in the pathogenesis of the hepatitis. In the present study, the mechanism of neoantigen generation was investigated. Liver microsomes from rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, +/- NADPH and/or NADH, were also analyzed. The results obtained demonstrate that neoantigen expression involves oxidative halothane metabolism by cytochromes P-450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially using the hapten derivative N-E-TFA-L-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus associated structural features of the protein carriers (100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa), not the TFA hapten alone. To our knowledge, this constitutes the first characterization of drug metabolite-tissue protein neoantigens implicated in a drug hypersensitivity. The approach described may be of general utility for characterization of drug-induced neoantigens associated with other drug hypersensitivities.     

A solid-phase radioimmunoassay for the measurement of lactoferrin in human plasma: variations with age, sex, and disease.

A solid-phase radioimmunoassay is described for measuring lactoferrin levels in normal human plasma. The sensitivity of the assay was 6 ng. per milliliter with an intraassay coefficient of variation of 4 per cent and an interassay value of 9 per cent. Healthy adult males had a mean plasma level of 1.62 mug per milliliter which was significantly higher than adult females, 1.07 mug per milliliter. Postmenopausal females had levels similar to men, 1.74 mug per milliliter, while younger women had a significantly lower mean value, 0.75 mug per milliliter. Two menstruating women and 2 pregnant women had moderately elevated levels. Consistently elevated levels were found in patients with untreated or relapsing chronic myeloid leukemia--all over 12.0 mug per milliliter, while patients on marrow suppressant therapy tended to have subnormal levels. The collection of serum specimens as opposed to plasma, resulted in inconsistently elevated levels: EDTA was the anticoagulant of choice, as heparin interfered in the radioimmunoassay system.     

Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: usefulness of enzyme-linked immunosorbent assay and enzyme immunotransfer blotting

The study reported here evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmunoelectrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the antibodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patient's sera recognize to-25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.     

The detection of IgG anti-hepatocyte antibodies by ELISA in sera of patients with chronic active hepatitis: correlation with disease activity

Anti-hepatocyte membrane IgG antibodies were detected in serial dilutions of sera from patients with chronic active hepatitis using an enzyme linked immunosorbent assay with isolated rat hepatocytes as the antigenic substrate. The assay is rapid, reliable and reproducible. Antibodies to hepatocyte membrane antigens were detected in 24 out of 31 (77%) patients with autoimmune chronic active hepatitis. Four of these patients were negative in a 1/10 dilution but became positive on progressive dilution. In 12 patients, liver biopsy was performed at the same time as serum was obtained. In these patients, the titre of antibodies to hepatocyte membrane antigen correlated significantly with the overall biopsy score of disease activity and particularly with the degree of portal tract infiltration. In 2 patients followed serially, titres of anti-hepatocyte membrane antibodies fell progressively in parallel with clinical, biochemical and histological evidence of improvement in the liver disease. The estimation of titres of antibodies to hepatocyte membrane antigens using isolated rat hepatocytes as the antigenic substrate may assist in the diagnosis, assessment of disease activity and follow-up of individual patients with chronic active hepatitis.     

Detection of antibodies to the E4 or E7 proteins of human papillomaviruses (HPV) in human sera by western blot analysis: type-specific reaction of anti-HPV 16 antibodies.

To determine the cross-reactivity between early (E) proteins of different human papillomavirus (HPV) types, 346 serum samples were tested with E4 and E7 of HPV 16. Two hundred and sixteen of them were also tested with HPV 1 E4, 21 with HPV 11 E4 and E7, and 109 with HPV 18 E4 and E7. Viral fusion proteins were expressed in Escherichia coli and used as antigens in Western blot experiments. The sera were obtained from patients with HPV-associated genital lesions or cervical cancer, from renal transplant recipients and from patients hospitalized for reasons unrelated to HPV infections (the controls). In contrast to findings relating to HPV 16 E4 specific antibodies, the prevalence of anti-HPV 1 E4 antibodies was not greater in renal transplant recipients than in the controls. In each age group of the control population more sera reacted with HPV 1 E4 than with HPV 16 E4. Sera of patients with HPV-associated cervical diseases and cervical cancer reacted less frequently with HPV 11 E4 or E7 and HPV 18 E4 or E7, respectively, than with the corresponding HPV 16 proteins. Thirty of 117 HPV 16 E4 or E7 positive sera showed reactivity to the corresponding protein of either HPV 1, 11 or 18. As demonstrated by cross-absorption experiments performed with 26 of the double-reacting sera, 24 contained two populations of antibodies reacting with proteins of different HPV types whereas only two contained cross-reacting antibodies. We concluded that in the majority of sera antibodies to the HPV 16 E4 and E7 proteins are type-specific.     

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto''s thyroiditis.

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.     

Importance of the detection method for thyroglobulin antibodies for the validity of thyroglobulin measurements in sera from patients with Graves disease.

BACKGROUND: The use of recovery tests has been proposed to disclose interferences from anti-thyroglobulin antibodies (TgAbs) in thyroglobulin (Tg) assays. We studied the value of a recovery test in Tg measurement by a new commercial IRMA. METHODS: Blood samples were collected from 153 patients with untreated Graves disease. Tg and TgAbs were measured by IRMA and RIA, respectively (Dynotest Tg-plus and Dynotest anti-Tgn; Brahms Diagnostica). The recoveries of added amounts of Tg were calculated for each serum. RESULTS: TgAbs were detected in 72 of the 153 patients (47%). The recovery test results for the 81 TgAb-negative sera (median, 101%; range, 80-115%) were identical to the results for the 91 controls (median, 102%; range, 80-124%). By contrast, significantly lower recovery test results were observed for the 72 TgAb-positive sera (median, 79%; range, 60-103%; Z = -8.363; P <0.0001). In the 34 of the 72 TgAb-positive sera with a normal recovery test, Tg concentrations were significantly lower (median Tg, 13.6 microg/L; range, 1.1-360 microg/L) than those measured in the TgAb-negative sera (median, 107 microg/L; range, 1.2-700 microg/L; Z = -3.797; P <0.0001). CONCLUSIONS: Tg values were decreased in TgAb-positive sera even when the results of the recovery tests were normal. This test should not be used alone to determine the validity of a serum Tg measurement in Graves disease.     

Measurement of cyclosporine in plasma from patients with various transplants: HPLC radioimmunoassay with a specific monoclonal antibody compared

This study compares cyclosporin A (CsA) concentrations in plasma from patients receiving various transplants, as measured by HPLC and RIA with a monoclonal antibody for CsA and an 125I-labeled ligand. The RIA was restandardized with in-house standards because it overestimated CsA by an average of 23%. The RIA was sensitive to 2 micrograms/L, the standard curve was linear from 20 to 500 micrograms of CsA per liter, analytical recovery was 98%, and CVs were less than 8% for intra- and interassay precision. RIA (y) vs HPLC (x) for 283 plasma samples from 145 patients gave a slope = 1.1256, r = 0.979. When the results were segregated according to transplant type, CsA in liver and heart recipients was overestimated by RIA as compared with HPLC: slope = 1.202, r = 0.973 and slope = 1.1477, r = 0.983, respectively. Adult and pediatric CsA values were acceptable when RIA and HPLC were compared: slope = 1.0755, r = 0.977 and slope = 1.0563, r = 0.980, respectively. For six samples (four heart, two liver recipients) where HPLC and RIA values demonstrated wide discrepancies, repeat HPLC and analysis of eluate fractions gave CsA concentrations nearer values by the initial HPLC assay. We conclude that this RIA cannot be substituted for HPLC in the case of heart and liver recipients. The need for each laboratory to standardize the RIA is obvious.     

Lymphocytotoxins in sera from highly sensitized multiparous dialysis patients: antibody class, relationship with the HLA and with paternal antigens.

1. Sera from 11 highly sensitized multiparous dialysis patients were studied in order to define the target antigens, antibody class and relationship with paternal HLA class I antigens of the underlying lymphocytotoxic antibodies. All sera contained lymphocytotoxic antibodies to over 70% of a panel of lymphocytes from 24 donors (panel reactivity greater than 70%). 2. Inhibition of cytotoxic activity against paternal lymphocytes by monoclonal antibodies to HLA framework determinants indicated that all 11 sera contained lymphocytotoxic antibodies to paternal class I antigens. In addition, five sera contained lymphocytotoxic antibodies to paternal class II antigens. 3. In order to determine the extent to which lymphocytotoxic antibodies were directed to paternal antigens, the panel reactivity of sera was compared before and after absorption with paternal peripheral blood lymphocytes. Over 50% of panel reactivity was absorbed from eight out of 11 sera, and in three of these 11 over 80% was absorbed. In the majority of patients this change in panel reactivity could be ascribed to binding of lymphocytotoxic antibodies to specific paternal class I antigens. 4. Digestion of sera with dithiothreitol had no significant effect on panel reactivity, indicating that the lymphocytotoxic antibodies were of immunoglobulin G class. 5. No sera reacted with either autologous lymphocytes or K562 cells, indicating an absence of autoantibodies. 6. These studies imply that panel-reactive lymphocytotoxic antibodies in the sera of highly sensitized multiparous patients are those which mediate hyperacute renal allograft rejection. Their development may be related to secondary humoral responses to antigens in blood transfusions from donors who share paternal class I specificities.     

Fibrin formation induced in agarose gel in the presence of plasma by sera from patients with certain diseases

During a period when screening for hepatitis B surface antigen (HBsAg) was performed by immunodiffusion using dextran-containing agarose gel, a diffuse precipitation (DP) zone was observed when citrate plasma samples were reacted with certain serum specimens. The DP reaction was noted with a significantly larger number of sera from patients with renal disorders, hepatitis, or certain other virus infections than with sera from apparently healthy blood donors, indicating that it was associated with some type of pathological condition. Highly purified fibrinogen used as detector reagent instead of plasma was sufficient to elicit a precipitation zone similar to that of the DP reaction. In the presence of coagulation inhibitors such as heparin, hirudin and antithrombin III the DP reaction was inhibited, suggesting that the precipitation zone represents coagulation. Cross-linked fibrin was demonstrated in the precipitates of DP-positive sera but not in the corresponding zone of a DP-negative serum.     

哈尔滨地区血小板输注无效患者的抗体鉴定与分析

目的探讨与分析哈尔滨地区免疫性血小板输注无效患者血小板抗体类型。方法对2017年11月-2018年6月的38例临床血小板输注无效的交叉配型阳性的患者,采用PAKPLUS试剂盒进行血小板特异性抗体鉴定,分析患者HLA抗体和HPA抗体分布情况。结果 36例患者出现抗体阳性,其中单一HLA-Ⅰ类抗体阳性5例(13.9%),单一HPA抗体阳性3例(8.3%),HLA-Ⅰ类抗体和HPA抗体均阳性28例(77.8%);2例未检出HLA-Ⅰ类抗体和HPA抗体。31例HPA抗体阳性中,患者血小板膜糖蛋白抗体鉴定结果为,GPⅡb/Ⅲa抗体26例(72.2%),GPⅠa/Ⅱa抗体23例(63.9%), GPⅠb/Ⅸ抗体19例(52.8%),GPⅣ抗体17例(47.2%)。抗体阳性患者中女性21例,男性15例;女性HLA抗体阳性率高于男性,女性HPA抗体阳性率低于男性,但差异无统计学意义。结论 PTR患者中血小板抗体以HLA-Ⅰ类抗体合并HPA抗体为主。     

Pregnancy specific beta 1 glycoprotein: a comparison of analysis by radioimmunoassay and enzyme linked immunosorbent assay observations on patients with malignant teratoma of testis

A comparison of serum pregnancy specific beta 1 glycoprotein analysis by enzyme linked immunosorbent assay and radioimmunoassay has been made. Excellent correlation was obtained between the two methods but radioimmunoassay was found to be more precise and to have a lower detection limit than enzyme linked immunosorbent assay. Using the more sensitive radioimmunoassay system, 2 out of 10 control male sera were found to contain detectable levels of pregnancy specific beta 1 glycoprotein. Sequential results are presented on patients with diagnosed malignant teratoma of testis who were undergoing chemotherapy.