A new assay for thyroglobulin concentration in serum using monoclonal antibodies against synthetic peptides

The concentration of thyroglobulin (Tg) measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) is greatly affected by the presence of anti-Tg autoantibodies in sera. We developed a new assay for detecting Tg in the presence of high concentrations of anti-Tg autoantibodies. A 48-kDa fragment was purified from Tg after treatment with V8 protease. This fragment did not appear to bind to two types of monoclonal antibodies (57Ab and 28D3) against a peptide in the C-terminus (amino acids 2735-2748) of Tg and intact Tg, respectively, by ELISA and Western blot analysis. In contrast, anti-Tg autoantibody or anti-Tg polyclonal antibody reacted well with this fragment. Our new ELISA used 57Ab as a solid phase antibody and 28D3 as a antibody conjugated to horseradish peroxidase. Buffer containing purified 48-kDa fragment was used to neutralize autoantibodies against Tg. With this assay, the recovery of Tg was 84.0-89.6% in normal healthy donors (n=5) in the presence of immunoglobulin G (IgG) purified from sera positive for anti-Tg autoantibody, and 76.2-104.4% in patient sera Grave's disease (n=15). Furthermore, the Tg concentrations in sera from patients with Grave's disease (n=20) ranged from 25 to 526 ng/ml, even though the Tg concentration, as measured by a commercial RIA did not exceed 55 ng/ml. There was good agreement between Tg concentrations measured by new Tg-ELISA and commercial Tg-RIA in sera that were negative for anti-Tg autoantibody. Overall, our new ELISA containing a Tg fragment to neutralize the presence of autoantibodies, showed good sensitivity and precision, and may be useful for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of thyroid autoimmunity or thyroid neoplasms.     

Medullary carcinoma of the thyroid

Summary This paper presents the results of the study of six patients with medullary thyroid carcinoma (MCT) and the epidemiological screening carried out on the families of individuals affected by MCT. Three patients had the familial and three the sporadic type of disease. In all the subjects the plasma calcitonin (CT) level was measured under basal conditions and after pentagastrin stimulation. Patients with familial disease were also found to have pheochromocytoma (MEA syndrome). One patient, whose thyroid was normal to palpation and radioisotope scanning and who did not have an elevated resting level of CT, showed a clearcut CT elevation after provocative testing and subsequently was shown, by surgery, to have a small nodule of C-cell hyperplasia. These results confirm that pentagastrin is a good stimulator of CT secretion and that i.v. administration of pentagastrin is a useful test in the investigation of MCT in its early subclinical stage.     

甲状腺髓样癌的诊断与鉴别诊断

目的探讨甲状腺髓样癌的形态特征、免疫表型、诊断与鉴别诊断。方法用免疫组化方法对25例甲状腺髓样癌进行研究,观察其病理形态特点。结果25例甲状腺髓样癌的癌组织主要由多角形及梭形细胞组成,可呈多种组织学类型,其中巢状型8例,束状/带状型6例,腺样(管状和腺泡)型3例,弥漫和类癌型各3例,透明细胞型和混合型(髓样和滤泡癌)各1例。免疫组化降钙素、铬粒素A均( ),21例神经元特异性烯醇化酶( ),21例癌胚抗原( ),18例突触素( ),1例HMB45( ),1例癌组织甲状腺球蛋白( )。CD44V6( )表达分别为髓样癌40%,滤泡癌30%,髓样癌80%颈部淋巴结转移,而正常甲状腺滤泡上皮(-)。结论诊断时应注意与形态相似的肿瘤相鉴别,CT是甲状腺髓样癌特异性标记物,若CT阴性,CEA及内分泌激素的阳性表达有助于甲状腺髓样癌的诊断和鉴别诊断。     

Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.     

Biological variation of thyroid autoantibodies and thyroglobulin.

BACKGROUND: It has been shown that the level of serum thyroid antibodies affects serum thyrotropin (TSH) concentrations in men and women, and that these autoantibodies in combination with serum TSH are predictive of future thyroid disease. As the biological variation of these autoantibodies is unknown, we investigated this in fertile women during one complete regular menstrual cycle. METHODS: A total of 24 healthy women (23-46 years) were investigated twice a week between 07:30 and 11:00 h. Antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb), and thyrotropin receptor (TRAb) were measured in serum, as well as thyroglobulin (Tg). TPOAb, TgAb and Tg were determined on an AutoDELFIA system (Perkin Elmer/Wallac) and TRAb by a radioreceptor assay from Brahms Diagnostica. RESULTS: All 24 women had measurable levels of TPOAb and TgAb in all samples, and nine women had antibodies above the upper reference limit of the laboratory (6 had TPOAb >10 kIU/L, 6 had TgAb >20 kIU/L and 1 had TRAb >0.75 IU/L). Eight women had Tg below the lower reference limit, five of whom had elevated TgAb. Variations in the thyroid antibodies were random and not related to the menstrual cycle. For TPOAb (2.5-258 kIU/L), the CV biological was 11.3%, while the CV analytical was 10.6%. For TgAb (5.6 to 148 kIU/L) CV biological was 8.5% and CV analytical was 9.0%. The woman with TRAb had a CV biological of 4.8%, while the analytical variation in duplicates was 3.9% at a level of 2.8 IU/L. CONCLUSIONS: It is possible to measure TPOAb and TgAb in all samples with the AutoDELFIA. There is no systematic variation in autoantibodies during the menstrual cycle. The biological coefficient of variation for TPOAb and TgAb was 11.3% and 8.5%, respectively.     

Long-term effects of thyroid fine-needle biopsy on the thyroid-related biochemical parameters

Aims: Thyroid fine‐needle biopsy (FNB) is a simple, reliable, inexpensive and generally safe diagnostic procedure in the management of thyroid nodules. FNB may trigger biochemical alterations through destruction of thyroid follicles. We aimed to investigate long‐term post‐FNB alterations in serum thyroid‐related parameters. Methods: One hundred and ten consecutive patients with thyroid nodular disease were subjected to FNB. Thyroid stimulating hormone (TSH), free thyroxine (FT4) and free triiodothyronine (FT3), thyroglobulin (Tg), thyroglobulin autoantibodies (anti‐Tg), thyroid‐peroxidase autoantibodies (anti‐TPO) were measured in all subjects at baseline, 10 days, 2 and 6 months. Subsequently, patients were divided into subgroups according to the technique of FNB, the presence of disease characteristics as thyroid autoimmunity (Hashimoto’s thyroiditis), goitre, singularity‐maximum diameter‐blood pattern of the nodule(‐s), the number of passes and the administration of L‐thyroxine (LT4). Results: A significant increase in Tg, anti‐Tg and FT3 levels was observed. These alterations were more prominent within patients with dominant nodule’s maximum diameter ≥ 2 cm or without Hashimoto’s thyroiditis. Tg and anti‐Tg levels were significantly increased only in patients not being on LT4. On the other hand, FNB technique did not affect any of the measured parameters. Conclusion: Our data suggest that FNB results in statistically significant but clinically insignificant increases in Tg, anti‐Tg and FT3 levels, implying a thyroid trauma of some level, more likely to happen in patients with larger nodules. The FNB technique used has no effect on the thyroid‐related biochemical parameters.     

Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to thyroglobulin

We used a computer programmed standard IgG curve for computer-assisted quantification of assay results for autoantibodies to thyroglobulin (Tg) by quantitative enzyme-linked immunosorbent assay (ELISA). Specific antibody levels in unknowns were quantified by comparison of their optical density readings with a standard curve of absorbance vs concentration obtained with dilutions of the reference serum. Anti-Tg antibodies were detected in 80% of the patients with chronic thyroiditis and 90% of those with Graves' disease. Anti-Tg antibodies were also detected in 14.3% of the healthy controls. The titer of anti-Tg antibodies detected by tanned red cell hemagglutination correlated well with that detected by ELISA, although, the sensitivity of the ELISA was higher. By our computer-assisted conversion method, the anti-Tg antibody can be readily and reliably quantified and low titer antibodies to Tg can be detected with adequate precision.     

Diagnosis of thrombotic thrombocytopenic purpura

As hallmark of TTP, generalized hyaline thrombi in the patient's microcirculation is known. These thrombi are composed of platelets and VWF. A severe defect of the VWF cleaving protease (VWF-CP) was found in all known patients with the inherited form of TTP. In contrary, although a severe deficiency of VWF-CP is specific for the acquired form, too, only a fraction of these patients is characterized by a severe deficiency. In most patients with a severe deficiency autoantibodies directed against VWF-CP is detectable in plasma. However, many patients with acquired TTP do not show any severe deficiency. Because treatment differs in inherited and acquired forms and as persistance of autoantibodies during clinical remission is of prognostic value, the determination of the activity of VWF-CP and of antibodies against VWF-CP are important parts in the workup of patients with TTP. In all methods for the determination of the activity of VWF-CP the first step is proteolysis of a specific substrate for the protease. In the second step the activity of the protease is measured by analysis of the residual VWF multimers, by the generation of specific fragments, by using the residual VWF:CB or VWF:RCo as marker of the loss of multimers or with help of specific monoclonal antibodies. In less than 30 min the cone and plate(let) aggregometer helps to distinguish between different forms of thrombotic microangiopathies. While adhesion and aggregation of platelets from a healthy person are clearly enhanced after addition of a small amount of plasma from a TTP patient, both characteristics are weakened by plasma from patients with other forms of thrombotic microangiopathy (dilution effect). Molecular genetics are established methods in the differentiation between inherited and acquired forms of TTP in those cases without autoantibodies against VWF-CP.     

Novel considerations on the antibody/autoantigen system in type I (insulin-dependent) diabetes mellitus

Insulin dependent diabetes (IDDM) has an autoimmune pathogenesis. Included is the presence of antibodies to pancreatic islet cells. The first identified were islet cell antibodies (ICA), detected by indirect immunofluorescence, and which react with all cells within islets. Importantly, the autoantibodies are found several years prior to disease and although a pathogenic role for the autoantibodies is unclear, they have become useful markers of prediabetes. A number of studies of twins discordant for IDDM and of first degree relatives of IDDM patients have established that there is an increased risk for disease in individuals who have ICA, especially when ICA levels are high. This high predictive value of ICA decreases in the general population where the incidence of IDDM is lower than in first degree relatives, and both ICA and the disease risk associated with ICA, appear to be influenced by a genetic susceptibility. This has been sustained in a study of patients with endocrine autoimmunity and ICA (Polyendocrine Study) where the predictive value of very high levels of ICA is less than 50% in patients without a first degree relative with IDDM. Hence, there remain a substantial number of patients with ICA who do not develop disease. From these patients, it was demonstrated that ICA include at least two distinct specificities, one of which is beta cell specific and is not associated with a high risk for IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen

Epidermolysis bullosa acquisita (EBA) is a subepidermal blistering disorder associated with tissue-bound and circulating autoantibodies specific to type VII collagen, a major constituent of the dermal-epidermal junction. Previous attempts to transfer the disease by injection of patient autoantibodies into mice have been unsuccessful. To study the pathogenic relevance of antibodies specific to type VII collagen in vivo, we generated and characterized rabbit antibodies specific to a murine form of this antigen and passively transferred them into adult nude, BALB/c, and C57BL/6 mice. Immune rabbit IgG bound to the lamina densa of murine skin and immunoblotted type VII collagen. Mice injected with purified IgG specific to type VII collagen, in contrast to control mice, developed subepidermal skin blisters, reproducing the human disease at the clinical, histological, electron microscopical, and immunopathological levels. Titers of rabbit IgG in the serum of mice correlated with the extent of the disease. F(ab')(2) fragments of rabbit IgG specific to type VII collagen were not pathogenic. When injected into C5-deficient mice, antibodies specific to type VII collagen failed to induce the disease, whereas C5-sufficient mice were susceptible to blister induction. This animal model for EBA should facilitate further dissection of the pathogenesis of this disease and development of new therapeutic strategies.     

Monoclonal immunoradiometric assay of calcitonin improves investigation of familial medullary thyroid carcinoma

Calcitonin (CT) assay is essential for recognizing medullary thyroid carcinoma (MTC), particularly occult familial MTC. In previous radioimmunoassays of calcitonin, polyclonal antibodies were used. Here we evaluate a new two-site immunoradiometric assay (IRMA) of calcitonin based on use of monoclonal antibodies. We assayed samples from healthy subjects, patients with renal failure, and subjects from families affected by MTC. Basal values for healthy subjects were all less than 10 ng/L. Renal failure is associated with increased basal CT. The CT peak under pentagastrin stimulation in healthy patients was less than 30 ng/L. In familial screening, basal values greater than 10 ng/L or peak values greater than 30 ng/L correspond to subjects with histologically confirmed MCT or micro-MCT. Polyclonal RIA performed in the same subjects failed to detect the moderate increase of CT that IRMA demonstrated. Preliminary results indicate that this new method may allow earlier detection of CT increase and thus improved diagnosis of MCT, particularly in familial screening. Monitoring surgical patients could also be improved by this new assay.     

Solving the problem of antibody interference in commercial "sandwich"-type immunoassays of carcinoembryonic antigen

We evaluated the effect of human anti-murine antibodies (HAMA) on apparent concentrations of carcinoembryonic antigen (CEA) as measured in serum with commercial enzyme immunoassay (EIA) kits manufactured by Abbot ("two-step" double monoclonal antibody assay), Roche, and Hybritech (room-temperature protocol). In sera from patients given parenteral murine monoclonal antibody for experimental diagnostic and immunotherapy studies, HAMA titers were determined with Immunomedics' "ImmuSTRIP HAMA-EIA" kit reagents. "True" CEA titers were established by using the ImmuCEA/MA-EIA and heat-extraction to destroy HAMA before assay for CEA. The concordance of the ImmuCEA/MA assay with the Abbott and Roche CEA EIAs was established with sera from normal individuals and from patients who had not received parenteral injections of murine monoclonal antibody. At high (100 mg/L) concentrations of HAMA, false-positive results were observed with all three kits. The Hybritech and Roche assays were more sensitive to interference by HAMA than was the Abbott CEA-EIA, false-positive results being observed at HAMA concentrations between 1 and 10 mg/L. Similar sensitivity of the three kits to interference by primate anti-MAb sera was demonstrated. Use of primate anti-MAb sera to create controls with HAMA activity and of analyte is recommended to evaluate MAb assays for potential HAMA interference and for use to devise methods to eliminate HAMA interference.     

Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I.

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.     

Novel Considerations on the Antibody/Autoantigen System in Type I (insulin-dependent) Diabetes Mellitus

Insulin dependent diabetes (IDDM) has an autoimmune pathogenesis. Included is the presence of antibodies to pancreatic islet cells. The first identified were islet cell antibodies (ICA), detected by indirect immunofluorescence, and which react with all cells within islets. Importantly, the autoantibodies are found several years prior to disease and although a pathogenic role for the autoantibodies is unclear, they have become useful markers of prediabetes. A number of studies of twins discordant for IDDM and of first degree relatives of IDDM patients have established that there is an increased risk for disease in individuals who have ICA, especially when ICA levels are high. This high predictive value of ICA decreases in the general population where the incidence of IDDM is lower than in first degree relatives, and both ICA and the disease risk associated with ICA, appear to be influenced by a genetic susceptibility. This has been substantiated in a study of patients with endocrine autoimmunity and ICA (Polyendocrine Study) where the predictive value of very high levels of ICA is less than 50% in patients without a first degree relative with IDDM. Hence, there remain a substantial number of patients with ICA who do not develop disease. From these patients, it was demonstrated that ICA include at least two distinct specificities, one of which is beta cell specific and is not associated with a high risk for IDDM.

These data, and an increasing list of new autoantibodies in IDDM, have increased the search for the relevant autoantigens. That of classic ICA is suggested to be a sialoganglioside, and a number of protein autoantigens have been suggested as other potential autoantigens. Of particular importance has been the identification of glutamic acid decarboxylase (GAD), the biosynthesizing enzyme of the neurotransmitter gamma amino butyric acid, as the autoantigen corresponding to the 64K autoantibodies found in the majority of newly diagnosed IDDM patients. Despite the identification of GAD, specific assays developed so far have not detected anti GAD antibodies at high frequencies in newly diagnosed patients. Like ICA, it appears that there may be distinct specificities of autoantibodies to GAD and associated products. Enzyme digestion of the autoantigen yields 50K, 40K and 37K fragments which appear to be derived from distinct polypeptide chains. Patients may have autoantibodies to either the 50K fragment or the 40/37K fragment or all fragments. Importantly, studies in twins and in the polyendocrine patients indicate that autoantibodies to the 37K fragment are more strongly associated with the progression to disease.

In addition to these autoantigens, autoantibodies are detected against the hormone insulin and its precursor proinsulin in up to 40% of newly diagnosed patients. Heat shock protein 65, carboxypeptidase H and glucose transporter have also been reported as autoantigens relevant to IDDM. It is now clear that the autoimmune response associated with IDDM is heterogeneous and against several islet antigens. The search will continue to determine which of these is important in the pathogenesis and which will provide effective markers for prediction which will eventually allow safe intervention therapy for the prevention of IDDM.     

Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. A specific marker of dihydralazine-induced hepatitis.

Sera from patients with dihydralazine-induced hepatitis were shown to contain anti-liver microsomal autoantibodies (anti-LM) by indirect immunofluorescence. These anti-LM antibodies were different from anti-liver/kidney microsomes (anti-LKM) 1 or 2 autoantibodies which have been previously described. Sera recognized a single 53,000 = Mr polypeptide in human liver microsomes as judged by immunoblotting, and the target antigen was identified as cytochrome P-450IA2 (P-450IA2) by (a) comparison of immunoblotting patterns with anti-human P-450IA2 and anti-rat P-450IA2 and with five anti-LM sera, and (b) specific immunoinhibition of microsomal ethoxyresorufin and phenacetin O-deethylation activities (both P-450IA2 supported reactions) by anti-LM antibodies. Finally, purified human P-450IA2 was recognized by these anti-LM sera. The anti-LM antibodies are specific for the disease because none of the other antisera tested behaved in the same manner as anti-LM, even those from patients treated with dihydralazine and without hepatic disease. A possible role of P-450IA2 in the metabolism of dihydralazine was suggested by competitive inhibition of ethoxyresorufin-O-deethylase observed in microsomal incubations. Thus, a new example is presented in which a cytochrome P-450 may be a target for autoantibodies in drug-induced hepatitis.     

人源抗CEA噬菌体抗体库的构建及筛选

目的构建人癌胚抗原(CEA)特异性噬菌体抗体库,制备人源抗CEA单克隆抗体(mAb)。方法用常规RT-PCR法,直接从10例结肠癌患者肠系膜淋巴结中的淋巴细胞克隆出免疫球蛋白Fd段和κ链基因,建成噬菌体抗体库。对抗体库进行5轮吸附-洗脱-扩增的亲和选择后,以ELISA法鉴定抗CEA噬菌体。结果RT-PCR可有效地扩增出Fd和κ基因并以此构建成容量为5.2×106的噬菌体抗体库。经5轮亲和选择可使特异性噬菌体抗体得到高度富集,并成功获得抗CEA噬菌体抗体阳性克隆。结论抗CEA噬菌体抗体库的构建和人源抗CEA mAb的制备,为CEA阳性表达肿瘤的靶向治疗奠定基础。     

Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies

A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs.     

Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine.

We have generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) designated 3H1, which mimics a specific epitope on the carcinoembryonic antigen (CEA). Patients with CEA positive tumors are immunologically "tolerant" to CEA. We used 3H1 as a surrogate for CEA for vaccine therapy of 12 patients with advanced colorectal cancer. Each of the patients received a minimum of four intracutaneous injections of aluminum hydroxide precipitated 3H1 at either 1, 2, or 4 mg dosage per injection. 9 of 12 patients demonstrated anti-anti-idiotypic (Ab3) response to 3H1. All nine patients generated specific anti-CEA antibody demonstrated by reactivity with radiolabeled purified CEA; some cases were confirmed by immunoprecipitation of purified CEA. We also demonstrated Ab3 stained both autologous and allogeneic colonic tumors. 7 of 12 patients demonstrated idiotype specific T cell proliferative responses and four also showed T cell proliferation to CEA. Toxicity was limited to local reaction with mild fever and chills. All 12 patients eventually progressed after finishing 4-13 dosages. This is the first report demonstrating that a vaccine therapy is capable of breaking "immune tolerance" to CEA in patients with CEA positive tumors. Future studies will focus on treating patients with minimal residual disease.