Solid-phase Monoclonal Antibody Research Articles

New insights into heparin binding to vitronectin: studies with monoclonal antibodies.

Vitronectin is a plasma glycoprotein that binds to a variety of ligands. There is considerable debate regarding the dependency of these binding interactions upon the conformational status of vitronectin, the role of multimerization and how the binding of different ligands can change vitronectin's conformational state. We have developed a method of capturing vitronectin directly from fresh plasma using solid-phase monoclonal antibodies. Various biotin-labelled secondary monoclonal antibodies were used to quantify the bound vitronectin and to measure its degree of denaturation. Using these tools we demonstrated that one monoclonal antibody partially denatured vitronectin without direct multimerization. Treatment of vitronectin in plasma with soluble heparin produced a similar degree of denaturation. These results led to a proposed adaptation of the unfolding/refolding pathways for chemically denatured vitronectin originally presented by Zhuang and co-workers in 1996 [Zhuang, Blackburn and Peterson (1996) J. Biol. Chem. 271, 14323-14332 and Zhuang, Li, Williams, Wagner, Seiffert and Peterson (1996) J. Biol. Chem. 271, 14333-14343]. The adapted version allows for the production of a more stable partially unfolded intermediate, resulting from the binding of particular ligands. We also demonstrated that the avidity of heparin binding to vitronectin is governed by both the conformational state of the monomer and multimerization of the molecule.

A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase

We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 μg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function. We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein.—Kobori, K., K. Saito, S. Ito, K. Kotani, M. Manabe, and T. Kanno. A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase.

One‐step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP‐20)

A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP-20, enamelysin) was established by use of a solid-phase monoclonal antibody and a separate peroxidase-labeled monoclonal antibody. The EIA system was shown to be sensitive and quantitative for the detection of MMP-20. As little as 1.0 ng/ml (50 pg/assay) of MMP-20 protein could be reliably detected. The EIA system was linear over a range of 2.5-160 ng/ml (125-8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP-20, active MMP-20, and MMP-20 with COOH-terminal deletions. The EIA system was validated by the successful detection of MMP-20 in the culture medium of Chinese hamster ovary cells (CHO-K1) that were transfected with an MMP-20 expression vector. No MMP-20 was detected in normal human serum, normal saliva, or in selected tumors. However, when recombinant human MMP-20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP-20, indicating that the system will work within the framework of complex in biological fluids.

Quantitation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes.

We raised monoclonal antibodies against human macrophage migration inhibitory factor (MIF), and developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) method highly specific for human MIF. The ELISA system utilizes a solid phase monoclonal antibody as a capture antibody and a horseradish peroxidase-conjugated monoclonal antibody as a detector antibody. We used this ELISA method to evaluate the serum level of MIF in 240 healthy volunteers (140 males and 100 females). We found no significant difference in MIF concentration with respect to age. A significant difference was found with respect to sex, with the mean value (+/- SD) for male subjects of 5.3+/-2.3, and that for female subjects of 4.6+/-2.3 ng/ml (p<0.05). We next measured the serum MIF contents of patients with autoimmune diseases, and found that MIF levels were significantly elevated in patients with systemic lupus erythematosus and rheumatoid arthritis, 20.0+/-11.0 ng/ml and 21. 7+/-11.2 ng/ml, respectively. Using anti-MIF antibody-immobilized sepharose column chromatography, we discovered for the first time that MIF was present in erythrocytes. Taken together these results suggest that MIF plays a major role in autoimmune diseases and, moreover, potentially induces various patho-logical outcomes in cases of hemolytic disorders.

Quality control of coated antibodies: new, rapid determination of binding affinity.

A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.

Injury to autologous normal tissues and tumors mediated by lymphokine-activated killer (LAK) cells generated in vitro from peripheral blood mononuclear cells of glioblastoma patients.

Activation of peripheral blood mononuclear cells (PBMC) with IL-2 generates lymphokine-activated killer (LAK) cells that show a broad target cell range. In adoptive immunotherapy using in vitro-generated LAK cells, the intensity and specificity of their cytotoxic activity affect the prognosis of cancer patients. The present study was designed to examine the tumor-specific spectrum of T lymphocytes generated from the PBMC of patients with recurrent glioblastoma by in vitro propagation with IL-2 plus either soluble or solid-phase anti-CD3 monoclonal antibody (MAb) in short-term or long-term cultures. Both short-term and long-term culturing with solid-phase anti-CD3 MAb plus IL-2 yielded broad-reactivity CD8+ alphabetaT and gammadeltaT lymphocytes, both of which were non-MHC restricted, as shown by the fact that they were able to lyse autologous glioblastoma cells, MHC class I+II- allogeneic glioblastoma cells, and MHC class I-II-NK-sensitive K562 target cells. More importantly, these cells from patients failed to lyse fresh autologous PBMC. These results demonstrate that cells generated using this approach are non-MHC-restricted LAK cells and exhibit marked tumor specificity. In contrast, incubation with soluble anti-CD3 MAb generated T lymphocytes that after long-term culture, were either CD4+ or CD8+. These caused significant lysis of both allogeneic and autologous glioblastoma target cells, the extent of lysis being greater than that using cells produced by culturing with the solid-phase MAb. However, both the CD4+ and CD8+ cells also caused greater lysis of autologous normal PBMC, indicating that cells generated using this approach may cause significant adverse reactions in cancer patients if used for immunotherapy.

An immunoradiometric assay for thymosin alpha 1.

An Immunoradiometric assay for thymosin alpha 1(1-28) was developed using a monoclonal and a polyclonal antiserum which were raised against synthetic thymosin alpha 1(1-28) and thymosin alpha 1(16-28), respectively. A monoclonal antibody, specific for the (acetylated ser1) 1-5 sequence of thymosin alpha 1, was immobilized on polystyrene beads for the solid phase, and a polyclonal antiserum specific for the 16-28 sequence was employed. This method relies on the formation of an immune complex consisting of a 125I-labelled anti-rabbit IgG goat antibody, polyclonal antiserum, thymosin alpha 1, and the solid phase monoclonal antibody. Radioactivity on the solid phase is directly proportional to the amount of thymosin alpha 1 present in the specimen. The minimal detection limit of this assay system was approximately 1.9 pg/ml. The mean values of thymosin alpha 1 in plasma of healthy subject, ranging in age from 0 to 3 years was approximately five fold higher than that of higher ages. HPLC analysis of plasma of a healthy subjects revealed a single immunoreactive form which eluted with the same retention time as that of synthetic thymosin alpha 1. This assay will be extremely useful for the measurement of thymosin alpha 1 in biologic fluids and tissues.

One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies

C4b-binding protein (C4BP), a regulatory component in the complement system, binds to an anticoagulant vitamin K-dependent plasma protein S (PS) which acts as a cofactor of activated protein C. We raised monoclonal antibodies against C4BP and PS, and developed two different one-step sandwich enzyme immunoassay (EIA) systems for human total C4BP (assay A) and PS-C4BP complex (assay B) by using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab′). The reaction time of the assay was 45 min in both EIA systems: 30 min for the immunoreaction and 15 min for the color reaction. The sensitivities were 12 and 20 mg/l in assays A and B, respectively. Linearity was obtained between 31 and 500 mg/l in both EIA systems. Assay A could detect both uncomplexed C4BP and PS-C4BP complex with equal efficiency so that totatl C4BP level was not affected by PS. The levels of total C4BP and PS-C4BP complex were found to significantly increase in sera from patients with membranous nephropathy and decrease with liver cirrhosis in comparison with the levels in normal subjects. On the other hand, a difference in the total C4BP and PS-C4BP complex levels was not shown between IgA nephropathy and normal subjects. Affinity column analysis and difference of total C4BP and PS-C4BP complex levels showed that most of C4BP in sera exists as PS-C4BP complex.

Direct chemiluminescence immuoassay of estriol and progresterone and their ratio during pregnancy

Estriol (E3) and progesterone (P) concentrations in saliva were determined by direct chemiluminescence immunoassays, using solid-phase monoclonal antibodies bound to the wells of microtitre plates, and isoluminol-labelled steroids conjugates. Saliva samples were obtained from women during pregnancy, from 34 to 1 weeks before delivery. The smoothed median and mean salivary E3:P ratios in normal pregnancy rose gradually from 0.45 at -34 weeks to 1.5 at -1 week. In twin pregnancy a similar rise was observed. However, in preterm delivery the smoothed mean ratio rose more slowly, from 0.6 at -30 weeks to 1.1 at -1 week. In pregnancy associated with intra-uterine growth retardation, the smoothed mean ratio did not reach a value of 1 towards the end of pregnancy. In all instances high and low E3:P ratios were observed shortly before spontaneous delivery. This raises the question of whether the E3:P ratio could be used as a predictor providing useful information in relation to the onset of labour.

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies

Interstitial collagenase (EC 3.4.24.7, matrix metalloproteinase-1, MMP-1) is synthesized and secreted by many cells, and plays an important role in a wide variety of pathophysiological degradation processes of extracellular matrices. The activity of MMP-1 is regulated by tissue inhibitors of metalloproteinases, TIMP-1 or TIMP-2, which form a non-covalent complex with the active enzyme. We raised monoclonal antibodies against zymogen of MMP-1, proMMP-1 purified from human skin fibroblasts. The antibodies recognized both precursor and active forms of MMP-1, but did not cross-react with 72-kDa and 92-kDa gelatinase/type IV collagenases or stromelysin-1. A specific and sensitive one-step sandwich enzyme immunoassay for human MMP-1 was developed using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The assay can be completed within 1 h (30 min for immunoreaction and 15 min for color development) and the sensitivity is 0.12μg/1 with the linearity between 0.12 and 10μg/l. Active MMP-1 shows 1.3-fold higher absorption at 492 nm than proMMP-1. However, the recognition rate of MMP-1 is decreased to ∼50% and <3% for the MMP-1-TIMP-1 and MMP-1-TIMP-2 complex forms, respectively. The MMP-1 levels in human sera from 120 healthy subjects are shown to be in the range of 8.5 ± 5.2 μ g/l ( mean ± S.D.) and the levels of 95% of the samples range from 0 to 20 μg/l.

A one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases-2 using monoclonal antibodies

A one-step sandwich enzyme immunoassay system was developed with a pair of monoclonal antibodies against two individual oligopeptides prepared from the amino acid sequence of the human tissue inhibitor of metalloproteinases-2 (TIMP-2). The assay system consisting of two simultaneous immunoreactions used a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody. The system detected a free form of TIMP-2 and that complexed with active forms of matrix metalloproteinases (MMPs) giving a different sensitivity for each MMP but not TIMP-2 complexed with the precursor of 72 kDa gelatinase/type IV collagenase (MMP-2). The sensitivity of the system was 1.6 μg/l (16 pg/assay) and linearity was obtained between 6.3 and 50 μg/l (63–500 pg/assay). TIMP-2 levels in the sera of 20 patients with rheumatoid arthritis (68 ± 25 μgl, mean ± S.D.) and 13 patients with hepatocellular carcinoma (76 ± 46 μg/l) were significantly higher ( P < 0.05) than those of 18 normal subjects (5.6 ± 7.4 μg/l). In contrast, the levels in the sera of 10 patients with gastric cancer (45 ± 18 μg/l) and 7 patients with cancer of the uterus (36 ± 13 μg/l) were significantly lower ( P < 0.05 or P< 0.01) than those of normal subjects. Immunoreactivity analyses suggested that the precursor of MMP-2 in normal sera exists in a complexed form with TIMP-2 by interacting with the C-terminal domain of TIMP-2.

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies

A one-step sandwich enzyme immunoassay (EIA) for matrix metalloproteinase 3 (MMP-3; stromelysin-1) was developed. The assay system used two simultaneous immunoreactions using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The sensitivity of the assay system was 20 μ/l and linearity was obtained between 31 and 500 μg/l. The EIA system was capable of measuring both precursor and active forms of MMP-3 as well as the forms of MMP-3 complexed with tissue inhibitors of metalloproteinases. MMP-3 levels as measured by this assay are significantly higher in the sera of patients with rheumatoid arthritis as compared to those of healthy subjects and patients with osteoarthritis. Immunoblot analyses showed that in the sera and synovial fluids of patients with rheumatoid arthritis, MMP-3 is present in the 59- and 57-kDa precursor forms.

Ultrasensitive Thyrotropin Immunoassay Based on Enzymatically Amplified Time-Resolved Fluorescence with a Terbium Chelate

We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.

C1q-bearing immune complexes detected by a monoclonal antibody to human C1q in rheumatoid arthritis sera and synovial fluids

Using a monoclonal antibody directed against the C-chain of human C1q, we detected C1q-bearing immune complexes (IC) in sera and synovial fluids of rheumatoid arthritis (RA) patients. In a sandwich-ELISA, C1q-bearing IC were captured by the solid-phase monoclonal antibody and then detected with peroxidase-labeled F(ab')2-antibodies to either human IgG or IgM. The results of this assay were compared to an ELISA-modification of the C1q-solid-phase binding assay (C1q-SPBA). C1q-bearing IC were detected in 81.1% of RA-sera and the 65.2% of RA-synovial fluids. IgG as well as IgM was present in 72.6% of the sera and 70% of the synovial fluids which were positive in both assays. Most RA sera that were only positive for C1q-bearing IC, contained IgG alone (81.5%). The corresponding synovial fluids showed IgG alone (53%) or both IgG and IgM (41.1%). IgM alone (25%) could be detected in sera, e.g. in juvenile forms of RA. The levels of IC were higher in synovial fluid than in paired serum. In comparison to normal human serum (NHS) and patients with osteoarthritis, complement activity (CH50 titers) and C1q-values in patients with RA were frequently elevated. Since the formation of C1q-bearing IC is an indicator for the classical complement pathway activation, an assay with monoclonal anti-C1q antibody may be a useful tool in the diagnosis of rheumatoid diseases.

Complement-Mediated Inactivation of Interferon-Gamma in Elisa Systems

The recovery of a predetermined amount of interferon-gamma (IFN-gamma) added to normal serum was studied in two independent sandwich ELISA systems specific for rat and human IFN-gamma. In both assays the ELISA activity was rapidly lost in fresh but not in heat-inactivated (30', 56 degrees C) serum. Ninety percent of the initial activity had disappeared within 30 minutes upon incubation at 37 degrees C. Serum-mediated inhibition was not species-specific as the ELISA activity of rat IFN-gamma diminished equally well in rat and human sera. Inhibition was critically dependent on the isotype of the solid-phase monoclonal antibody (mAb) used in the ELISA systems. IgG1 and IgG2a mAbs efficiently inhibited the ELISA activity of IFN-gamma, whereas an IgA mAb was ineffective. The inhibition was not influenced by a wide variety of anti-proteolytic agents but was effectively blocked by anti-complementary substances or treatments directed to the first (C1) and third (C3) component of complement. Our results indicate that activation of the classical pathway of complement (CPC) and the concomitant covalent binding of C3 to the IFN-gamma molecule play a major role in the inhibitory process. It is concluded that reduction of the ELISA activity is attributable to diminished accessibility of the detector antibody for the IFN-gamma protein as a consequence of C3 binding.

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells.

An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Solid-phase monoclonal antibodies A novel method of directing the function of biologically active molecules by presenting a specific orientation

Monoclonal antibodies were produced against fibronectin and vitronectin, using the extracellular matrix from cultured bovine corneal endothelial cells as immunogen. Some antibodies inhibited the adhesion of BHK-21 cells to these molecules, in a standard assay with fibronectin or vitronectin coated on a plastic surface. By first coating the plastic surface with the monoclonal antibodies and subsequently binding vitronectin, the inhibitory effect was markedly increased. Divalent fibronectin required the presence of antibody in the medium as well as coated on the surface for maximal inhibition of cell adhesion. The inhibitory effect of surface-coated antibodies was stable in the presence of cells up to at least 24 h in vitro. Using this antibody coating method the surface could either be made compatible for cell adhesion (using non-inhibitory antibodies) or refractory. Assays performed by this method have considerable potential for quantifying particular functions of extracellular matrix molecules.

Time-Resolved Immunofluorometric Assay for Thyroxine-Binding Globulin in Serum

We describe a new "sandwich"-type immunofluorometric assay for thyroxine-binding globulin (TBG) in serum. The assay involves a solid-phase monoclonal antibody immobilised in white microtiter wells, and a soluble biotinylated monoclonal antibody that reacts with the captured TBG molecules. Addition of streptavidin labeled with the europium chelator, BCPDA (4,7-bis(chlorosulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid), and excess europium results in the formation of a highly fluorescent product. The fluorescence signal of the final complex is quantitated on the dried solid-phase with a pulsed-laser time-resolved fluorometer. The assay requires a 151-fold sample pre-dilution and a total incubation time of 90 minutes. It has a broad dynamic range of 0-100 mg/L and a minimum detection limit of 0.4 mg/L. The coefficients of variation for within-run and between-run assays averaged 4.5% and 5.4%, respectively. The mean analytical recovery of TBG added to serum was 103%. Results obtained by this method correlated well with those determined by a commercial radioimmunoassay (r = 0.96, n = 112) and by an immunoradiometric procedure (r = 0.95, n = 131).

Direct Photolabeling of the cGMP-stimulated Cyclic Nucleotide Phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal antibodies

Six mouse monoclonal antibodies against carcinoembryonic antigen were evaluated for use in a solid-phase immunometric assay. Three IgG2 antibodies, when attached to polymer particles or microtiter wells, were found to be severely inhibited by fresh serum. The remaining three antibodies, which were of the IgG1 subclass, were inhibited only slightly or not at all when used in the same way. With the aid of labelled antibodies against C1q and C3, it was shown that antibody inhibition was accompanied by the binding of large amounts of these complement factors. Experiments involving heat inactivation or dilutions of serum, or the addition of EDTA, consistently revealed the same correlation between binding of complement factors and inhibition of CEA binding to antibody. It is suggested that the significant inhibition of the solid-phase IgG2 antibodies was caused by classical pathway activation of complement by the solid-phase antibody even in the absence of antigen. The slight inhibition of solid-phase IgG1 antibodies observed at high serum concentrations was probably due to complement binding by the alternative pathway. Preliminary evidence suggests that complement interference is not restricted to CEA assays, but is a potential problem in all types of serum immunoassays using solid-phase antibodies.