Cerebrospinal fluid levels of neurofilament light in chronic experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) is a chronic relapsing-remitting animal model of multiple sclerosis (MS). Neurofilament light (NF-L), a structural protein expressed in neuronal cells can be used to quantify the amount of neuronal damage in MS patients. An immunoassay was used to measure levels of neurofilament light in cerebrospinal fluid (CSF) in rats with myelin oligodendrocyte glycoprotein-induced EAE. Significantly increased levels of neurofilament were found in the immunized animals compared to the controls, strengthening the similarities in the diseases and the progression pattern between the animal model and MS. The turnover of NF-L during this disease is increased since significantly elevated levels also were identified in the spinal cord of the diseased animals and immunohistochemistry gave support for this observation. Monitoring neurofilament levels in EAE can be used to follow disease progression and effects of therapy.

Neurofilament and glial fibrillary acidic protein in multiple sclerosis.

OBJECTIVE: To evaluate levels of neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) in CSF from patients with multiple sclerosis (MS) in relation to clinical progress of the disease. METHODS: CSF levels of NFL and GFAP were determined by sensitive ELISAs in 99 patients with different subtypes of MS, classified in terms of "ongoing relapse" or "clinically stable disease," and 25 control subjects. Levels were compared with paraclinical data such as immunoglobulin G index and inflammatory cell count in the CSF, and the levels were related to Expanded Disability Status Scale score and progression index at clinical follow-up evaluations later in the disease course. RESULTS: NFL and GFAP levels were elevated in MS patients as compared with control subjects (p < 0.001). The NFL levels were higher at relapses, whereas GFAP levels were unaffected. High NFL levels correlated with progression in patients with an active relapse (r = 0.49; p < 0.01) and in clinically stable patients (r = 0.29; p < 0.05). GFAP correlated to progression in the total patient cohort (r = 0.24; p < 0.05). Moreover, a strong correlation between NFL levels and inflammatory cell counts was evident in the group of patients with an ongoing relapse (r = 0.52; p = 0.001). CONCLUSIONS: CSF levels of neurofilament light and glial fibrillary acidic protein may have prognostic value in multiple sclerosis.

Interfering with cancer: a brief outline of advances in RNA interference in oncology.

RNA interference (RNAi) is a potent and ubiquitous gene-silencing mechanism that is generating considerable excitement in the fields of molecular biology and gene therapy. It is now in widespread use for loss-of-function analysis in many diseases including cancer. Nevertheless, RNAi is still in its infancy, with new discoveries appearing on a monthly basis. This article presents a brief outline of the history and recent advances in RNAi with a specific focus on its potential in oncology.

Tumor marker workshops.

Since 1996, the nine ISOBM Workshops have so far characterized more than 300 monoclonal antibodies to a variety of tumor markers that include CA125, AFP, PSA, MUC1, Cytokeratins, Sialyl Le(a), hCG, CEA, ALP, and more recently SCC, and S100. Besides the basic characterization of antibodies and their epitope configurations, several workshops have also addressed specific problems associated with multiple antigen variants. These workshops have been able to make significant advances well beyond those possible through any normal collaboration study. The data and impact of these workshops with their summary reports are reviewed.

Native and transformed alpha2-macroglobulin in plasma from patients with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease with unknown etiology. Various proteinases have been observed in increased levels in the central nervous system of patients with MS, which may contribute to the release of immunogenic myelin components. alpha2-Macroglobulin (alpha2M) inhibits a broad spectrum of proteinases sterically, undergoing major conformational changes induced by the proteinases themselves. Moreover, alpha2M acts as a carrier of several cytokines in the systemic circulation. By use of radial immunodiffusion, we determined the total alpha2M levels in plasma from 28 MS patients and 15 control subjects [14 patients with other neurologic diseases (OND) and one healthy individual]. No significant differences in total alpha2M concentration were observed between the MS patients and the control subjects. A comparison of the degree of alpha2M transformation in MS patients with different disease courses and controls was performed, using monoclonal antibodies (mAbs) specific for binding to native and transformed alpha2M, respectively. The fractions of transformed alpha2M were significantly increased in patients with secondary or primary progressive disease course compared with the controls. No significant differences were obtained using a native-specific mAb. At least a major proportion of alpha2M from the MS patients was able to change conformation from its native to its transformed state, as demonstrated by a shift in mAb reactivity, following methylamine treatment of the plasma samples. In conclusion, the results indicate that plasma alpha2M may be inactivated at a higher degree in patients with chronic progressive MS compared with patients with OND. This may influence the levels of proteinases and cytokines in the systemic circulation and may furthermore have diagnostic implications.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

Apoptosis in HeLa Hep2 cells is induced by low-dose, low-dose-rate radiation.

Radioimmunotherapy with radiolabeled antibodies may cause inhibition of the growth of epithelial tumors, despite low total radiation doses and comparatively low radiosensitivity of epithelial tumor cells. The induction of apoptosis by low-dose radiation, such as delivered in radioimmunotherapy, was investigated in vitro in human HeLa Hep2 carcinoma cells. The cultured cells were exposed to defined radiation doses from a (60)Co radiation therapy source. The radiation source delivered 0.80 +/- 0.032 (mean +/- SD) Gy/min and the cells were given total doses of 1, 2, 5, 10 and 15 Gy. Using fluorescein-labeled Annexin V, followed by flow cytometry and DNA ladder analysis, apoptotic cells were detected and quantified. Radiation doses below 2 Gy did not cause any significant increase in apoptosis. Compared to control cells, apoptosis was pronounced after 5-10 Gy irradiation and was correlated to the radiation dose, with up to 42 +/- 3.5% of the cells examined displaying apoptosis. Clonogenic assays confirmed significantly decreased viability of the cells in the interval 2 to 10 Gy with low-dose-rate radiation, 60 +/- 2% compared to 2 +/- 2%. Lethal effects on the tumor cells were also evaluated by an assay of the cytotoxic effects of the release of (51)Cr. Significant cytotoxicity, with up to 64 +/- 6% dead cells, was observed at 5 Gy. Similar results were obtained when the dose rate was reduced to 0.072 +/- 0.003 Gy/min (mean +/- SD). In the case of the (137)Cs source, the dose rate could be reduced to 0.045 Gy/h, a level comparable to radioimmunotherapy, which induced significant apoptosis, and was most pronounced at 72-168 h postirradiation. It can be concluded that in vitro low-dose and low-dose-rate radiation induces apoptosis in epithelial HeLa Hep2 cells and thus may explain a mechanism by which pronounced inhibition of growth of HeLa Hep2 tumors at doses used in radioimmunotherapy has been obtained previously.

Radioimmunoscintigraphy using an anti-prostate monoclonal antibody (E4): a dosimetric evaluation.

The aim of this study was to evaluate different strategies to increase the tumour radiation dose for experimental radioimmunotherapy using 125I-labelled monoclonal antibody (MAb) E4 in a nude mice model xenografted with DU-145 tumours. The effects from a single injection of the 125I-labelled MAb E4, the same total amount of radiolabelled MAb E4 divided into three repeated injections, and the effect of pre-targeting with non-labelled MAb E4 for reducing the amount of shed antigen were investigated. Based on repetitive quantitative radioimmunoscintigraphies, calculation of the tumour radiation dose delivered from the 125I-nuclide was performed for each strategy. The single injection strategy without pretargeting rendered the highest mean tumour radiation dose, i.e. 0.23 Gy/MBq. Pretargeting with non-labelled MAb E4 before a single injection of [125I]E4 resulted in a slightly lower mean tumour radiation dose, i.e. 0.19 Gy/MBq, compared to the single injection alone. An even lower mean tumour radiation dose, i.e. 0.14 Gy/MBq, was obtained when the same total administered amount of activity was divided into three separate injections given in 10-day intervals. We concluded that the single injection strategy is the most efficient when using MAb E4 in this tumour model. The tumour radiation doses were not increased by dividing the same amount of activity into three injections or by pretargeting with non-labelled MAb E4.

Conformational state and receptor recognition of the C-terminal domain of human alpha(2)-macroglobulin after dissociation into half-molecules.

BACKGROUND: Dissociation of native human alpha(2)-macroglobulin (alpha(2)M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated alpha(2)-macroglobulin. METHODS: The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of alpha(2)-macroglobulin. RESULTS: The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed alpha(2)-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of alpha(2)-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric alpha(2)-macroglobulin in competition studies. CONCLUSIONS: It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.

Structural evidence for a functional role of human tissue nonspecific alkaline phosphatase in bone mineralization.

The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Ménez, A., and Stura, E. A. (2001) J. Biol. Chem, 276, 9158-9165). The degree of homology allowed us to build a reliable TNAP model to investigate the relationship between mutations associated with hypophosphatasia and their probable consequences on the activity or the structure of the enzyme. The mutations are clustered within five crucial regions, namely the active site and its vicinity, the active site valley, the homodimer interface, the crown domain, and the metal-binding site. The crown domain and the metal-binding domain are mammalian-specific and were observed for the first time in the PLAP structure. The crown domain contains a collagen binding loop. A synchrotron radiation x-ray fluorescence study confirms that the metal in the metal-binding site is a calcium ion. Several severe mutations in TNAP occur around this calcium site, suggesting that calcium may be of critical importance for the TNAP function. The presence of this extra metal-binding site gives new insights on the controversial role observed for calcium.

Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity.

Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors. The crystal structure, determined at 1.8-A resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli. The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved. Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M. F., Manes, T., and Millán, J. L. (1997) J. Biol. Chem. 272, 22781-22787). Allostery is probably favored by the quality of the dimer interface, by a long N-terminal alpha-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other. In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by l-amino acids, consistent with the acquisition of substrate specificity. The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.

The versatility of cytokeratins as tumor markers.

Cytokeratins are particularly versatile tools in tumor biology both experimentally and clinically. Previously regarded as a rigid intracellular skeleton, the cytokeratins are now known to provide a more dynamic structural integrity within epithelial cells. This more flexible nature of cytokeratin function occurs through rapid shifts in assembly/disassembly states, geared by phosphorylations/dephosphorylations in the 'head' and 'tail' regions of the monomeric filaments. When released from proliferating or dying tumor cells, they provide a useful marker for epithelial malignancies, as evidenced by the number of available immunochemical assays for cytokeratins. Another significant area of interest that highlights their utility is the deposition of cytokeratins in the necrotic areas of tumors, providing an effective target for radioimmunotherapy and radioimmunolocalization. Moreover, the appearence of cytokeratin neodeterminants seems to be a very early event during apoptosis. Indeed, the involvement of specific cytokeratin degradation patterns resulting from the activity of caspases during apoptosis highlights yet another developing field of research devoted to these highly pleomorphic and complex structures.

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

Accumulation of(125)iodine labeled interleukin-2 in the pancreas of NOD mice.

Interleukin-2 (IL-2) is an important cytokine in the autoimmune process proceeding Type 1 diabetes. Our aim was to investigate, in two previously used animal models, the NOD mouse and the BB/W rat, the in vivo tissue distribution of radio-labeled IL-2. If the radio-labeled IL-2 accumulated significantly in the pancreas compared to surrounding organs it could allow imaging of lymphocyte infiltration of the islets of Langerhans by scintigraphic methods. IL-2 was labeled enzymatically with(125)Iodine. Radio-labeled IL-2 was injected iv in prediabetic NOD mice, diabetic NOD mice and Balb/c mice in the first animal model and in BB rats in the second model. Animals were sacrificed at different time points and the activity in different organs was measured. It was found that the mean activity in the pancreas in both diabetic and prediabetic NOD mice was significantly higher compared to pancreas from Balb/c mice (P< 0.001 and P=0.005, respectively). However, the mean activity in the pancreas was at the lower range of the surrounding organs in both animal models, thereby excluding the possibility of imaging the autoimmune process by scintigraphic methods. It is concluded that radio-labeled IL-2 did accumulate significantly in the pancreas of NOD mice compared to control mice but there is a need to develop new techniques in order to visualize the localized activity.

Immunochemical aberrations of alpha2-macroglobulin purified from a patient with multiple sclerosis.

We report on a patient with primary progressive multiple sclerosis who was found to present alpha2-macroglobulin with aberrant immunochemical properties. alpha2-macroglobulin was purified from plasma and investigated in ELISA using highly conformation specific monoclonal antibodies. The purified alpha2-macroglobulin displayed reactivity with antibodies specific for binding to native alpha2-macroglobulin as well as with antibodies specific for binding to transformed alpha2-macroglobulin. Furthermore, following methylamine treatment, alpha2-macroglobulin from the multiple sclerosis patient presented a reduced shift in antibody reactivity from the native-specific to the transformed-specific antibodies, as compared to normal alpha2-macroglobulin. In conclusion, the results suggest that alpha2-macroglobulin from this multiple sclerosis patient presents conformational aberrations, which may have implications on the capacity to eliminate proteinases from the systemic circulation.

Conformational variants of human alpha2-macroglobulin are reflected in a C-terminal 'switch region'.

Human alpha2-macroglobulin displays extensive conformational changes when induced to transform into new quaternary structures, which are eliminated from the systemic circulation by receptor-mediated endocytosis. One major region involved in these conformational changes is located in a segment of 30 amino acids from Glu1314 to Ser1343 (-Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Gln-Thr-Leu-Pro-Gln-Thr-Cys-Asp -Glu-Pro-Lys-Ala-His-Thr-Ser-Phe-Gln-Ile-Ser-Leu-Ser-), which we term the 'switch region' of alpha2-macroglobulin, as deduced by immunochemical techniques. Monoclonal antibodies were generated using either native, methylamine-treated or the 18-kDa C-terminal receptor-binding fragment as the immunogen. From an extensive number of obtained hybridomas, 11 mAbs were selected because of their capacity to bind to the C-terminal fragment. Irrespective of the original configuration of the antigen used for immunization, seven of the antibodies were shown to be reactive with a set of overlapping epitopes, closely positioned within the 'switch region', as confirmed by the use of synthetic peptides covering the entire C-terminal fragment. The specificities of the seven individual antibodies, as determined by ELISA and BIAcore technologies, revealed a pronounced conformational pleomorphism in the 'switch region'. The results indicate that the 'switch region' may be involved in the exposure of the receptor recognition site and can be used as an indicator region for different conformational states of alpha2-macroglobulin.

Aberrant forms of alpha(2)-macroglobulin purified from patients with multiple sclerosis.

The biochemical properties of alpha(2)-macroglobulin were investigated in four patients with multiple sclerosis and compared to alpha(2)-macroglobulin from healthy controls. An impaired stability of alpha(2)-macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic"fast" form of alpha(2)-macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of alpha(2)-macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between alpha(2)-macroglobulin from the multiple sclerosis patients and alpha(2)-macroglobulin from healthy controls. The results suggest that aberrant forms of alpha(2)-macroglobulin may be present in patients with multiple sclerosis.

Decreased stability of alpha2-macroglobulin purified from patients with multiple sclerosis.

OBJECTIVES: To investigate conformational properties of alpha2-macroglobulin from multiple sclerosis patients. MATERIALS AND METHODS: alpha2-macroglobulin was purified to homogeneity from plasma of 4 multiple sclerosis patients and 5 healthy controls. The plasma and the purified alpha2-macroglobulin from each individual were investigated using polyacrylamide gel electrophoresis. RESULTS: Impaired stability of purified alpha2-macroglobulins from multiple sclerosis patients was demonstrated with spontaneous conversion to an electrophoretic "fast" form upon purification and following storage not ascribable to bait region cleavage. CONCLUSION: alpha2-macroglobulin from multiple sclerosis patients displays altered stability. Possible functional impairments of proteinase inhibition mechanisms are discussed.

A new specific method for measuring S-100B in serum.

The S-100 family of proteins are acidic calcium and zinc binding low molecular weight proteins mainly present in astrocytes and in a population of oligodendrocytes of the CNS. S100b, an acidic low weight and zinc binding protein, has attracted considerable interest due to its release into the cerebrospinal fluid and blood from brain tissue following brain damage and from malignant melanomas. A new simple two-step incubation assay has now been elaborated in which two catcher and one tracer monoclonal antibodies are used. The specificity of this assay is high because all the MAbs used bind exclusively to S-100B, as shown by real-time biospecific interaction analyses. Moreover, the working range of the assay is 0.2-60 micrograms/L with a CV of less than 10%; the resulting high sensitivity has been confirmed by clinical studies. Time dependence, shaking conditions, lower limit of detection limits, effects of dilution, hook effect, recovery, impression as intra- and interassay variations, and crossreactivities with S-100A1 were tested in order to obtain a highly reproducible assay. Sera from healthy blood donors and patients undergoing cardiopulmonary bypass operations were tested with the assay. Several of the patients undergoing open heart surgery presented measurable values in this IRMA S-100 assay, indicating cerebral effects of open heart surgery. The test may be used for postoperative monitoring of these patients.

Idiotypic-anti-idiotypic antibody interactions in experimental radioimmunotargeting.

Idiotypic-anti-idiotypic antibody interactions can be used in vivo to regulate the serum levels of specific radiolabeled antibodies. Anti-idiotypic antibodies can also be used as clearing agents for radiolabeled antibodies in radioimmunolocalization and radioimmunotherapy. The present study describes the immunochemical interactions between the monoclonal idiotype (H7) and three generated monoclonal anti-idiotypic antibodies (alphaH7:1, alphaH7:35, and alphaH7:38). An unexpected variability in complex formation could be demonstrated in vitro, revealing three different stable complex patterns, i.e., low molecular weight 1:1 complexes, ladder formation with oligomeric, consecutively added constituents, and large linear polymeric complexes of high molecular weight. Within 24 h, the anti-idiotypes were able to cause a significant decrease in total body radioactivity, and the antibody generating a ladder formation (alphaH7:38) was found to be the most efficient at removing radiolabeled idiotypes from the circulation. It is concluded that monoclonal anti-idiotypic antibodies may be valuable tools in improving radioimmunolocalization and radioimmunotargeting.