Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGß, hCGßn, hCGßcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.

Alpha fetoprotein is increasing with age in ataxia-telangiectasia.

The elevated serum alpha fetoprotein (AFP) concentration in ataxia-telangiectasia (A-T) patients has been known for decades, but the individual variation of AFP levels over time has not been studied. We have followed 12 patients (five girls and seven boys) for 1-12 years (mean 5.5 years) measuring in each patient AFP 2-8 (mean 4) times. Serum AFP levels were increased in all patients, mean 168.7 (range 40-373) kU/L, and without significant differences between the patients. There was a significant age related difference in the serum AFP level. A positive linear relationship (r=0.61, p=0.04) could be found between AFP level and age. Albumin levels were within normal range and did not change with age. Four patients had slightly increased aspartate aminotransferase (AST) levels. None of the patients had serological evidence of infectious hepatitis, and none had increased levels of carcinoembryonic antigen. Repeated standardized observations of gait function revealed no major difference in neurological deterioration between our patients. All had classical A-T disease and mainly truncating mutations; 21 out of 24 possible mutations were either frameshift or nonsense. Four were homozygous for the Norwegian ATM founder mutation. No correlation between serum AFP levels and the different ATM genotypes could be found. We conclude that serum AFP is not only elevated, but also is continuously increasing with age in patients with classical A-T disease.

The usefulness of detecting thyroglobulin in fine-needle aspirates from patients with neck lesions using a sensitive thyroglobulin assay.

The aim of this study was to assess the diagnostic utility of thyroglobulin (Tg) in fine needle aspirates (Tg-FNAB) of nonthyroidal neck masses using a sensitive in-house method for detecting Tg in washout specimens. A total of 256 samples from 145 patients were evaluated for Tg in washout specimen from FNAB and compared to corresponding cytological smear and histology of 46 surgical specimens. Tg was measured by a sensitive in-house time-resolved immunofluorometric assay. The sensitivity for Tg-FNAB alone or in combination with cytological findings was found to be 100% in both the follow-up group and before primary surgery. In the follow-up group the specificity of Tg-FNAB was 100%. Fifty-nine of 60 follow-up specimens with malignant cytology were Tg-FNAB positive (n = 195). Histological examination of one lymph node with malignant cytology and negative Tg-FNAB showed metastasis from carcinoma of the salivary gland. Tg-FNAB was positive in 25 specimens with suspicious or cystic cytology. Tg-FNAB values were high (median 4557 microg/l, range 122-37200 microg/l) in washout specimen from cystic metastasis from which cytology did not confirm malignancy. Of the 20 lymph nodes with histology confirming metastasis from differentiated thyroid carcinoma (DTC), the Tg-FNAB was positive in 19 and intermediate in one. However, before primary surgery, two Tg-FNABs were false positive compared to the histology of the lymph nodes. TgAb in serum did not interfere with FNAB-Tg measurements. Tg-FNAB measurement is accurate with high sensitivity (100%) and of great importance in detecting cystic metastasis when cytology is not conclusive. Even metastases to small neck lymph nodes may be detected by using sensitive Tg-assay. Serum thyroglobulin antibodies appear to have ignorable effect on the clinical performance of Tg-FNAB.

Reaction of alpha-mannosidase from Phaseolus vulgaris with group-specific reagents. Essential carboxyl groups.

When the pKm of alpha-mannosidase was determined at different pH values, the results indicated that ionizable groups with pK values of approx. 3.8 and 5.7 could be essential. Modification with carbodiimide or Woodward's Reagent K abolished the enzyme activity. The substrate analogue, alpha-methyl-D-mannoside, protected the enzyme against inactivation. Incorporation of a 14C-labeled nucleophile reagent in the presence or absence of the analogue suggested that 2--4 carboxyl groups were protected. Exchange studies indicated that the essential Zn2+ could be bound to such groups. There was no indication that hydroxyl groups, sulphydryl groups, guanidino groups or amino groups take part in the catalytic activity.

The chemical modification of tryptophan residues of alpha-mannosidase from Phaseolus vulgaris.

Reaction of alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from Phaseolus vulgaris with N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide- resulted in loss of enzyme activity. Spectral absorption and fluorescence studies, as well as amino acid analysis, suggested that only tryptophan residues had been modified. No change in conformation could be detected by density gradient ultracentrifugation or circular dichroism of alpha-mannosidase modified by N-bromosuccinimide to virtually zero enzyme activity. The inhibition was partly offset by the substrate analogue alpha-methyl-D-mannoside and the competitive inhibitor mannono-1,4-lactone. Concomitantly, two tryptophan residues fewer were oxidized per molecule. After modification V was reduced, while Km seemed unchanged. Further, there was found evidence for the enzyme having a secondary structure dominated by beta-pleated sheets.

Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

Neuron-specific enolase as a prognostic factor in metastatic malignant melanoma.

Serum neuron-specific enolase (NSE) was measured in 63 patients with metastatic malignant melanoma. 20 patients (32%) had elevated serum NSE (> 10 micrograms/l) before the start of treatment. Another 13 patients (21%) developed pathological NSE values during the course of the disease. In many patients, elevated NSE was related to a large tumour burden, and a gradual rise in serum NSE indicated disease progression. Patients with elevated pretreatment NSE had a median survival time of 3 months compared with 12 months for those with normal pretreatment NSE values. NSE thus proved to be a useful prognostic factor in metastatic malignant melanoma.

Dual analyte assay based on particle types of different size measured by flow cytometry.

Simultaneous flow cytometric assays have been developed for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), with internal determination of sample related non-specific binding (NSB). The assays use particles of 7.5, 6.5 and 5.5 microns diameter coated with, respectively, monoclonal antibodies specific for AFP, hCG or an epitope normally not present in serum. The different particle types were identified simultaneously by light-scatter measurements as their specific immunofluorometric responses were determined. The NSB in the simultaneous assay of AFP and hCG was increased by approximately 30% compared to corresponding single analyte assays. The working range of the dual analyte assays was 0.6-2000 kIU/l for AFP and 6-10,000 IU/l for hCG. No significant interference from the presence of the other analyte was observed in the measurement of either AFP or hCG. The 95% confidence interval for the ratio of dual over single analyte assay results was [0.81, 1.11] for AFP and [0.88, 1.16] for hCG.

Neuron specific enolase (NSE) in serum of patients with malignant melanoma.

Serial measurements of serum NSE were performed in 63 patients with metastatic melanoma. NSE was measured by a sensitive immunoradiometric assay based upon monoclonal anti-bodies with monodisperse magnetizable particles as the solid phase. Increased NSE values were found in 30 patients (48%) during the course of the disease. In 10 patients serum NSE normalized during systemic therapy. In 11 patients with initial normal serum NSE, the marker values became elevated as the tumor progressed.

Prognosis in patients with metastatic non-seminomatous testicular cancer.

155 patients with metastatic non-seminomatous testicular cancer were treated with cisplatin-based chemotherapy which in most cases was combined with surgery. The 5 year crude survival was 90% for all patients (98 patients with small volume disease: 97%; 32 patients with large volume disease: 91%; 25 patients with very large volume disease: 64%). High pre-chemotherapy serum tumour marker levels (AFP greater than 500 micrograms/l; and/or HCG greater than 1000 U/l) decreased the survival rates in all groups. Only 4 of 17 relapsing patients were rendered tumour-free by salvage chemotherapy. In a multivariate analysis, a pre-chemotherapy alpha-foetoprotein (AFP) level greater than 500 micrograms/l was associated with poor survival as was the presence of a retroperitoneal tumour greater than 10 cm, lung metastases greater than 3 cm and/or extrapulmonary hematogenous metastases. It is concluded that easily assessable clinical pre-treatment variables can be used to define high risk or low risk patients with metastatic testicular cancer. Treatment intensity should be adjusted in accordance to such prognostic factors.

A pharmacokinetic evaluation of IM administration of bleomycin oil suspension.

Bleomycin oil suspension was given IM twice daily to four patients, and bleomycin saline solution infused to three patients with cervical carcinoma. The serum levels of bleomycin were followed for 12 h by radioimmunoassay. Both regimens revealed comparable side effects. Only minor responses were seen. Bleomycin oil suspension produced prolonged levels of bleomycin in serum.

An enzyme antigen immunoassay for the determination of neuron-specific enolase in serum samples.

A method is presented for the detection and quantification of neuron-specific enolase (NSE) in serum samples. It is an enzyme antigen immunoassay (EAIA), relying on specific antibodies to 'catch' the enzyme on a solid support (ELISA-plate) whereafter the enzymatic activity of the immunocaptured enzyme is determined, by coupling the reaction to lactate dehydrogenase. The oxidation of NADH to NAD is followed at 340 nm in an ELISA photometer. The method is proven to work well and is able to measure the low amounts of NSE present in serum samples from normal individuals. It does not require labelling of neither antigen nor antibody, and is therefore superior to the commercially available radioimmunoassay (RIA). The method will also work with monoclonal antibodies towards the enzyme as demonstrated by preliminary observations.

Whole-body distribution of radioactivity after intraperitoneal administration of 32P colloids.

The whole-body distribution of radioactivity after intraperitoneal instillation of 32P-labelled chromic hydroxide particles has been studied in patients operated for early-stage ovarian cancer. Gamma-camera imaging of the abdominal 32P-distribution revealed that the administration procedure was critical for obtaining a homogeneous plating of the radiocolloids on the serosal surface. Dose calculations based on a uniform distribution of 32P in a capillary layer covering the intraperitoneal surface gave an estimated tissue surface dose of about 30 Gy per 370 MBq of 32P administered. The amount of 32P in peripheral blood increased for seven days after instillation followed by a continuous decrease. Bone marrow concentration was from two to five times as high as that in blood, but the total amounts were too small to give significant radiation doses. Gel chromatography showed that 33% of the activity in blood consisted of high molecular weight material, probably colloids. The remainder of the activity (67%) was attached to material of very low molecular weight, appearing as a consequence of physiological degradation of the colloids.

Thyroglobulin in patients with differentiated thyroid carcinoma.

Thyroglobulin (Tg) is a sensitive marker for detection of metastatic disease in patients who have been operated on for differentiated thyroid carcinoma. A high serum Tg value during thyroxine suppression therapy is indicative of metastatic disease, but the sensitivity is enhanced by the endogenous TSH response occurring when thyroxine medication is withheld. False-negative values before thyroxine withdrawal can be demonstrated in about 20% of the patients in our material. If not all thyroid tissue has been eradicated the interpretation of the serum Tg can be difficult. Tg cannot be used to differentiate benign from malignant disease prior to surgery.

Increased cerebrospinal fluid levels of nerve cell biomarkers in narcolepsy with cataplexy.

BACKGROUND: The association between narcolepsy with cataplexy and the hypocretinergic system in the central nervous system is strong since up to 75-90% of all patients have cerebrospinal fluid (CSF) hypocretin-1 deficiency. The predominant occurrence of HLADQB1*0602 tissue type in narcolepsy patients and recent results from genome-wide association studies suggest an underlying immunological mechanism. The present study was initiated to clarify whether measurement of nerve cell biomarkers in CSF could give additional knowledge of the pathophysiological mechanisms causing narcolepsy with cataplexy. METHODS: Two patient groups with narcolepsy, comprising 18 patients with low CSF hypocretin-1 concentrations and typical cataplexy, and 18 patients with normal CSF hypocretin-1 levels and mild cataplexy-like symptoms, were compared to 17 controls. We measured the nerve cell biomarkers beta-amyloid (Aß42), total tau protein (T-tau), phosphorylated tau (P-tau) and neuron-specific enolase (NSE) in CSF. RESULTS: The concentrations of all biomarkers were significantly elevated in both patient groups compared to the controls. The concentration of beta-amyloid was significantly higher in the patient group with normal CSF hypocretin-1 concentration than in those with low concentrations, whereas the other biomarkers showed no difference between the patient groups. CONCLUSION: The findings of elevated levels of CSF biomarkers independent of CSF hypocretin-1 reduction may reflect alterations in cell metabolism. The results suggest a more extensive affection of the sleep regulating cellular network, affecting other neuronal sites important in the regulation of sleep, in addition to the hypocretin-producing neurons.

alpha-Mannosidase from Phaseolus vulgaris. Composition and structural properties.

Both alpha-mannosidases I and II from Phaseolus vulgaris have molecular weights about 210000-220000 and contain approximately 2 mol zinc/mol protein. alpha-Mannosidase I seems to consist of more glutamic acid than alpha-mannosidase II, while the latter is richer in serine. They are glycoproteins: alpha-mannosidase I contains 8.3% carbohydrate by weight while alpha-mannosidase II contains 16.5%. This enzyme form shows a greater thermal stability than alpha-mannosidase I. The structure of alpha-mannosidase has been investigated by equilibrium sedimentation analysis in guanidine hydrochloride, electrophoresis in dodecylsulphate, and alkaline electrophoresis after exposure to high pH. The protein appears to be composed of two non-covalently bound subunits of molecular weights about 110000. Electron micrographs revealed images of molecules that consisted of two rod-shaped monomers of roughly square cross-sections 4.2 X 4.2 nm. Each rod was about 7.4 nm long. The monomers seemed parallell along the long axis.

Establishment and evaluation of a radioimmunoassay for neuron-specific enolase. A marker for small cell lung cancer.

Neuron-specific enolase (NSE) was purified from human brain to a specific activity of 91 U/mg with no demonstrable impurities. The enzyme has a molecular weight of 96,000, consists of two subunits of 47,000, and has an isoelectric point at pH 4.5. A radioimmunoassay based on antibody raised in sheep was established. The assay has a sensitivity of 2 micrograms/l, and an interassay coefficient of variation of 6.4% at 10 micrograms/l. The reference limit was defined as 10 micrograms/l, as found in sera from a population of 389 persons. Of 50 untreated patients with small cell lung cancer (SCLC), 70% had NSE levels above 10 micrograms/l. None of 170 patients with non-SCLC neoplasms had elevated enzyme levels.