Anticardiolipin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface.

Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.

High-density lipoprotein inversely relates to its specific autoantibody favoring oxidation in thrombotic primary antiphospholipid syndrome.

Abnormalities of the lipid profile partly explain the atherogenic tendency of systemic lupus erythematosus but the picture is unclear in thrombotic primary antiphospholipid syndrome (PAPS). Herein we compare the lipid profile, high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein A (ApoA-I), apolipoprotein B (ApoB), triglycerides (TRY)), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-ss(2)GPI) and C-reactive protein (CRP) from thrombotic PAPS (n = 34), thrombotic patients with inherited thrombophilia (IT; n = 36), subjects persistently positive for antiphospholipid antibodies (aPL, n = 18) with no underlying autoimmune or non-autoimmune disorders and healthy controls (n = 28) and determined the reciprocal effects of anti-lipoprotein antibodies, the lipid profile, oxLDL-ss(2)GPI and CRP. Average concentrations of HDL (p < 0.0001), LDL (p < 0.0001), CHO (p = 0.0002), ApoA-I (p = 0.002) were lower in PAPS whereas average TRY was higher (p = 0.01) than other groups. Moreover, the aPL and PAPS group showed higher levels of IgG anti-HDL (p = 0.01) and IgG anti-ApoA-I (p < 0.0001) whereas the PAPS group showed greater average oxLDL-ss(2)GPI (p = 0.001) and CRP (p = 0.003). Within the PAPS group, IgG anti-HDL correlated negatively to HDL (p = 0.004) and was an independent predictor of oxLDL-ss2GPI (p = 0.009). HDL and ApoA-I correlated negatively with CRP (p = 0.001 and p = 0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favoring low-grade inflammation and enhanced oxidation in thrombotic PAPS.

Pathophysiology of beta2-glycoprotein I in antiphospholipid syndrome.

Since beta(2)-glycoprotein I (beta(2)GPI) was described as the major antigenic target for antiphospholipid antibodies, many studies have focused their attention to the physiological role of beta(2)GPI and anti-beta(2)GPI antibodies on autoimmune-mediated thrombosis. Studies reporting the physiological role of beta(2)GPI have been numerous, but the exact mechanism of action(s) has yet to be completely determined. beta(2)GPI's epitopes for anti-beta(2)GPI autoantibodies have been characterized, however, not all of the heterogeneous anti-beta(2)GPI antibodies are pathogenic. The pathophysiologic role of beta(2)GPI has been reported in the fields of coagulation, fibrinolysis, angiogenesis, and atherosclerosis. Our understanding of the impact of beta(2)GPI, its metabolites and autoantibodies to beta(2)GPI on these physiological functions may contribute to the development of better therapeutic strategies to treat and prevent autoimmune-mediated atherothrombotic vascular disease.

Regulation of cellular immunity prevents Helicobacter pylori-induced atherosclerosis.

Helicobacter pylori (H. pylori) is a predominant pathogen that causes not only gastroduodenal diseases but also extra-alimentary tract diseases. In this study, we demonstrated that H. pylori infection promoted atherogenesis in heterozygous apoe(+/ --) ldlr(+/--) mice. The male mice were fed with high fat diet from the age of 6 weeks. At the age of 16 weeks, development of atherosclerotic lesions was observed in the H. pylori-infected mice, and it seemed to be associated with an elevation of Th1-immune response against H. pylori origin-heat shock protein 60 (Hp-HSP60) and an increment of transendothelial migration of T cells. Subcutaneous immunisation with Hp-HSP60 or H. pylori eradication with antibiotics significantly reduced the progression of atherosclerosis, accompanied by a decline of Th1 differentiation and reduction of their chemotaxis beyond the endothelium. Thus, oral infection with H. pylori accelerates atherosclerosis in mice and the active immunisation with Hp-HSP60 or the eradication of H. pylori with antibiotics can moderate/prevent cellular immunity, resulting in a reduction of atherosclerosis.

[Post-infectious clinical-immunological syndrome and its place in clinical practice].

The progression of chronic-relapsing infectious disease (CRID) depends on a combination of cumulative immune-mediated responses of the human body, which, in turn, are united by a number of the common mechanisms. The mechanisms are called as the post-infectious clinical-immunological syndrome (PICIS) to demonstrate the features and scale of imbalances of immune homeostasis. PICIS usually accompanies most of the known CRID to define the type of the disease, to predict the progression of and risks for the complications to be risen as well. PIFIS is generally provoked by either infectious pathogens of various nature or by the atypical immune responses from the infected patient, or by the onset of the disease itself, or by the inadequate antimicrobial therapy. Three forms of PICIS which depend on two key factors have been described. These included: (i) the spectrum of a microbial colonization landscape; (ii) the antimicrobial immunity itself to generate, for instance, either of three alternative PICIS, namely, (1) postinfectious secondary immunodeficiency syndrome (PISIS); (2) postinfectious autoimmune syndrome (PIAIS), and (3) PISIS combined with PIAIS, i.e. PISIDAS. The dominant monosyndrome-like form of associated immune imbalances in CRID patients is PISIS. PISIS occurs in more than a third of the clinical cases to stress the autoaggression (PIFAS), or combininative form of the immune-mediated imbalances, i.e. PISIDAS. In the process of the development of CRID, PISIS can give a way to either PIAIS or PISIDAS. Besides the immune-mediated imbalances, an essential role in the pathogenesis of CRID and PICIS is also attributed to the infectious factors capable of forming microbial associates in the pathogenesis of PICIS. Therefore, treatment of such patients should be directed not only at the elimination of the infectious pathogen(s), but also at the restoration of the physiological level of the immune homeostasis impaired by PICIS.

Primary antiphospholipid syndrome: a low-grade auto-inflammatory disease?

OBJECTIVE: To test the inflammation and immune activation hypothesis in primary thrombotic APS (PAPS) and to identify clinical and laboratory factors related to inflammation and immune activation. METHODS: PAPS (n = 41) patients were compared with patients with inherited thrombophilia (IT, n = 44) and controls (CTR, n = 39). IgG aCL, IgG anti-beta2-glycoprotein I (beta(2)GPI), high-sensitivity CRP (hs-CRP), serum amyloid A (SAA), CRP bound to oxidized low-density lipoprotein-beta(2)GPI complex (CRP-oxLDL-beta(2)GPI) (as inflammatory markers) neopterin (NPT), soluble CD14 (sCD14) (as immune activation markers) were measured by ELISA. RESULTS: After correction for confounders, PAPS showed higher plasma levels of hs-CRP (P = 0.0004), SAA (P < 0.01), CRP-oxLDL-beta(2)GPI (P = 0.0004), NPT (P < 0.0001) and sCD14 (P = 0.007) than IT and CTR. Two regression models were applied to the PAPS group: in the first, IgG aCL and IgG beta(2)GPI were included amongst the independent variables and in the second they were excluded. In the first model, SAA (as the dependent variable) independently related to thrombosis number (P = 0.003); NPT (as the dependent variable) independently related to thrombosis type (arterial, P = 0.03) and number (P = 0.04); sCD14 (as the dependent variable) independently related to IgG beta(2)GPI (P = 0.0001), age (0.001) and arterial thrombosis (P = 0.01); CRP-oxLDL-beta(2)GPI (as the dependent variable) independently related to IgG beta(2)GPI (P = 0.0001). In the second model, sCD14 and NPT independently related to each other (P = 0.002) (this was noted also in the IT group, P < 0.0001) and CRP-oxLDL-beta(2)GPI independently predicted SAA (P = 0.002). CONCLUSION: Low-grade inflammation and immune activation occur in thrombotic PAPS and relate to clinical features and aPL levels.

Autoimmune diseases and infections: controversial issues.

The etiology and pathogenesis of certain types of disease remain controversial and stand like a bridge that crosses infectious, autoimmune and autoinflammatory pathways. Infection, for example, may initiate a disease, although it is the genetic regulation in the host, the interplay between virus or bacteria persistence and autoimmunity that produces the later phases of disease, the antigenic determinants responsible for inducing autoimmune disease, and the pathogenetic effector mechanisms. Infections agents cause pericarditis, but in 85% of cases it is "idiopathic". It has also been shown that persistent Clamydia pneumoniae, Porphyromonas gingivalis, and Helicobacter pylori infections cause host immunity and promote atherogenesis. A number of infectious agents have been suggested as potential triggers for primary biliary cirrhosis. Infections and vaccinations have also been linked to the pathogenesis of fibromyalgia syndrome, a common, chronic syndrome of widespread pain. Many factors are also responsible for fever of unknown origin such as: infections, autoimmunity disease, etc. However, it is difficult to determine a direct correlation between the infections agents in such a large group of diseases. The aim of this review is to analyze some of the controversies about the role of infections in autoimmune diseases.

[Architectonics of cell subpopulations of peripheral blood in patients with autoimmune myocarditis: clinical and pathogenetic aspects].

AIM: To compare the most significant architectonic parameters of peripheral blood cell subpopulations in patients with different variants of an autoimmune myocarditis (AIM) course and their clinical value in therapeutic practice. MATERIAL AND METHODS: Blood cell subpopulations were studied with flow cytometry in 99 blood samples from patients having different AIM variants and myocardiosclerosis as well as in 40 healthy donors. RESULTS: Severe (malignant) AIM was characterized by growing indices of T-/B lymphocyte activation, expression of activation markers on the cells of both differentiation lines, disproportions in composition of subpopulations of the immunoregulatory cells, parallel rise in specific weight of dendritic cells, reduced intensity of apoptosis of autoreactive T-lymphocytes. In benign AIM marked immunopathology was not found. This group can be considered as a separate variant of AIM course necessitating an individual approach to planning pathogenetically sound therapeutic and rehabilitation measures. CONCLUSION: The study of activation markers expression on peripheral blood cells is superior to the study of endomyocardial biopsies providing a non-invasive method of immunodiagnosis.

Anti-beta2-glycoprotein I (beta2GPI) monoclonal antibodies with lupus anticoagulant-like activity enhance the beta2GPI binding to phospholipids.

beta2-Glycoprotein I (beta2GPI), a plasma glycoprotein with phospholipid-binding property, is known to be the actual target antigen for autoimmune type anticardiolipin antibodies (aCLs). Certain groups of aCLs (anti-beta2GPI antibodies) exert lupus anticoagulant (LA) activity and perturb the function of vascular endothelial cells. This investigation aimed at highlighting some insights into the molecular basis by which aCLs exert their biological effects by using anti-beta2GPI mAbs with well-characterized epitopes from mice and from patients with antiphospholipid syndrome. Anti-beta2GPI mAbs directed against the third domain (Cof-20 and Cof-22) and fourth domain (Cof-21, EY1C8, and EY2C9) of beta2GPI inhibited the thrombin generation induced by Russell's viper venom in diluted plasma and that induced by the prothrombinase complex reconstituted with purified clotting factors. This anticoagulant activity was abrogated in the presence of an excess amount of phospholipids, thus resembling the LA activity. In stark contrast, anti-beta2GPI mAbs directed against the fifth domain and the carboxy-terminal region of the fourth domain showed no LA-like activity. These findings suggest that the LA activity of anti-beta2GPI antibodies depends on their epitope specificity. Experiments carried out to clarify the mechanism of the LA activity showed that anti-beta2GPI mAbs with LA-like activity, but not those without this effect, enhance the beta2GPI binding to phospholipids. In addition, the F(ab')2 fragment, but not the Fab' fragment, of the anti-beta2GPI mAbs was found to enhance the LA activity and the beta2GPI binding to phospholipids, suggesting that anti-beta2GPI antibodies induce formation of multiple complexes of beta2GPI on the surface of phospholipids because of their bivalent property. This clustering of beta2GPI molecules induced by anti-beta2GPI antibodies, probably because of their multivalent property and epitope specificity, might hinder the lateral mobility and activation of clotting factors on the surface of phospholipids and thus exert LA activity. Clustering of beta2GPI molecules may also explain the molecular mechanism by which anti-beta2GPI antibodies alter the function of leukocytes and endothelial cells. The well-documented heterogeneous LA activity of aCLs (anti-beta2GPI antibodies) may also be explained by their epitope specificity.

Cancer-associated immune-mediated syndromes: Pathogenic values and clinical implementation.

The ability of tumors to provoke formation of cancer-associated secondary immunodeficiency (CASID) with predominant suppression of CMI and cancer-associated secondary immunodeficiency with clinical autoimmunity syndrome (CASICAS) with triggering of a set of the autoimmune deviations is appearing to be a key event in the restriction of hosts' anti-tumor immunity. Earlier the existence of the above-mentioned syndromes was demonstrated in BCC and GBM patients. In order to reach a point where immunological phenotypes in GBM and BCC can be clarified clinically and, partly, pathogenically, we have conducted a series of studies of typical and atypical types of immune responsiveness in patients with GBM and BCC. For GBM and BCC three scenarios of the involvement of the immune responsiveness have been established in a series of our studies, i.e., (i) malignancy with no immunopathology, (ii) malignancy as CASID, and (iii) malignancy as CASICAS. All of those scenarios demonstrated significant differences in their immune-mediated manifestations which, in turn, were proven to reveal close associative relationships with a specific clinicopathologic type and clinical manifestations of the tumor. CASID and CASICAS share two common features, i.e., (i) signs of immunodeficiency and (ii) a tandem of the deviations within the adaptive and innate links of the host immune responsiveness. At the same time, CASID and CASICAS are distinct pathogenically and clinically, and in terms of depth of the immune deviations observed, CASID patients manifest a breakage in both links, whereas in CASICAS patients, a breakage in the adaptive link would dominate. To get these differences clarified, we summarized major types of the immune imbalances and sets of clinical and clinicopathologic manifestations to illustrate the above-mentioned features in CASID and CASICAS patients. There are distinct close correlations between clinicopathologic features of the disease course and sets of the immune-mediated imbalances in patients harboring the tumors. The latter implicates a panel of the new immunodiagnostic and immunoprognostic criteria for patients with solid tumors, i.e., BCC, MCC and GB, which is of great value for clinical practice. In particular, the blood levels of some of the immunocompetent cells, state of their functional activity, serum titers of the antigenic markers and autoantibodies, apoptotic parameters, and others may be accepted as additional and clinically informative criteria to be implemented for immunological monitoring and immunotherapy of patients with solid tumors.

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine.

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids".

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.

Induction of mitochondrial DNA heteroplasmy by intra- and interspecific transplantation of germ plasm in Drosophila.

A new experimental system for inducing mitochondrial DNA heteroplasmy in Drosophila was developed. By transplanting the germ plasm of Drosophila melanogaster and Drosophila mauritiana into the posterior pole of the recipient eggs of D. melanogaster, it was possible to introduce foreign mitochondria into the recipient female germline. Heteroplasmic individuals containing both donor and recipient mtDNA were obtained in intra- and interspecific combinations at similar frequencies. The proportion of donor-derived mtDNA in the heteroplasmic individuals varied considerably from individual to individual irrespective of the donor species used. No significant decrease in or elimination of donor mtDNA was observed, and the heteroplasmic state in female germlines persisted for several generations. The present system should serve very much to promote the study and clarification of the transmission genetics of mtDNA in insects.

Incomplete maternal transmission of mitochondrial DNA in Drosophila.

The possibility of incomplete maternal transmission of mitochondrial DNA (mtDNA) in Drosophila, previously suggested by the presence of heteroplasmy, was examined by intra- and interspecific backcrosses of Drosophila simulans and its closest relative, Drosophila mauritiana. mtDNAs of offspring in these crosses were characterized by Southern hybridization with two alpha-32P-labeled probes that are specific to paternal mtDNAs. This method could detect as little as 0.03% paternal mtDNA, if present, in a sample. Among 331 lines that had been backcrossed for ten generations, four lines from the interspecific cross D. simulans (female) x D. mauritiana (male) showed clear evidence for paternal leakage of mtDNA. In three of these the maternal type was completely replaced while the fourth was heteroplasmic. Since in this experiment the total number of fertilization is known to be 331 x 10 = 3310, the proportion of paternal mtDNA per fertilization was estimated as about 0.1%. The mechanisms and evolutionary significance for paternal leakage are discussed in light of this finding.

Changes in the rat liver mitochondrial DNA upon aging.

During experiments on the molecular basis of morphological and functional changes observed in rat liver mitochondria upon aging, we found that the buoyant density profile of mitochondrial DNA (mtDNA) shows a wide distribution pattern especially in the lighter region than that of young rat liver mtDNA. The heterogeneous pattern may be partly recovered to become similar to that of young rat liver mtDNA by treatment with proteinase K. Therefore, it is quite likely that mtDNA of old rat liver contains firmly bound protein(s) or peptides. During the morphological observation of mtDNA by electron microscopy, we found that mtDNA of old rat had a novel property, that is, the ability to attach to negatively charged mica in the absence of magnesium ions, although their morphological features showing circular 5 microns contour length form did not change. Further, mtDNA gained resistance against EcoRI digestion during aging. This property was not shared by the DNA from young animal, and might be due to the binding protein(s).

Autosomal dominant familial spinal and bulbar muscular atrophy with gynecomastia.

The proband, a 53-year-old man, developed progressive spinal and bulbar muscular atrophy and gynecomastia at the age of 50. His father had weakness of lower limbs, and his son had a nasal voice, ocular movement abnormalities, and gynecomastia, whereas two of the proband's brothers showed either gynecomastia or tongue fasciculations. None of the patients showed any expansion of CAG repeat in the androgen receptor gene or any hormonal abnormality. Thus, this family is affected by a form of autosomal dominant spinal and bulbar muscular atrophy with gynecomastia.

Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 are increased in POEMS syndrome.

The authors quantitatively measured levels of matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP), and vascular endothelial growth factor (VEGF) in blood samples of POEMS syndrome. Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 were more increased in patients with POEMS syndrome than in patients with other neurologic disorders or in healthy controls. Serum levels of VEGF and TIMP-1 were strongly correlated with each other. Increased circulating levels of MMP-1, -2, -3, -9, and TIMP-1 may lead to a better understanding the pathogenesis of POEMS syndrome.

beta(2)-glycoprotein I deficiency: prevalence, genetic background and effects on plasma lipoprotein metabolism and hemostasis.

beta(2)-glycoprotein I (beta(2)-GPI=apolipoprotein H) is an important autoantigen in patients with the antiphospholipid syndrome. It also plays a role in lipoprotein metabolism, such as anti-atherogenic property, triglyceride removal, and enhancement of lipoprotein lipase. Serum beta(2)-GPI concentration of 812 apparently healthy Japanese individuals was measured by sandwich EIA. Two families with complete beta(2)-GPI deficiency were identified. In one family, all affected had increased serum LDL-cholesterol levels or smaller particle sizes of LDL, while the other had no apparent abnormality in lipid metabolism. Individuals investigated had no history of thrombosis or overt abnormalities in hemostatic tests. A thymine corresponding to position 379 of the beta(2)-GPI cDNA was deleted in every beta(2)-GPI deficient individual. The incidence of this heterozygous deficiency determined by RFLP was 6. 3% in Japanese and none in Caucasians. Heterozygotes had significantly lower concentrations of serum beta(2)-GPI than did those without the mutation, yet no significantly different lipid profiles, such as total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, apoA-I, apoB and Lp(a), were observed. A low concentration of beta(2)-GPI seemed not to be associated with apparent abnormality in lipoprotein metabolism.

Enzyme-linked immunosorbent assay for human pulmonary surfactant protein D.

We developed a simple and sensitive enzyme-linked immunosorbent assay (ELISA) for human pulmonary surfactant protein D (SP-D). Human SP-D was purified from bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis. Nine monoclonal antibodies (MAbs) were established from BALB/c mice immunized with the purified human SP-D. All MAbs were directed to either 43 kDa SP-D contained in lung lavage fluids of patients with pulmonary alveolar proteinosis or in amniotic fluids from healthy normal pregnancies. The ELISA is based on a sandwich method using two MAbs, 6B2 and 7C6. Cross-reactivity to human SP-A or rat SP-D was evaluated as below 0.6%. The recovery of different concentrations of SP-D ranged from 94.4% to 111.2%, and serial dilutions of amniotic fluids showed good linearity. SP-D concentrations in 21 amniotic fluids from normal pregnancies were measured by the ELISA. The mean concentration in amniotic fluids from pregnancies in the third trimester was significantly higher than that from earlier stages of gestation (p < 0.001), indicating that this ELISA may be applicable for prediction of fetal lung maturity.

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3).

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.