A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies of defined specificity. I. Application to naturally occurring antipolyamine antibodies in human sera.

A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies with a defined specificity. II. Application to immune complexes containing viral antigens in human sera.

The coupling of viral antigens from parainfluenza virus (PIV-1), cytomegalovirus (CMV) and human immunodeficiency virus (HIV-1) to chemically functionalized polystyrene plates has permitted us to develop a covalent enzyme-linked immunoassay (CELIA) for measuring the titers of free antibody (Ab) and immune complex (IC) bound Ab directed against each of these viruses. The method was first validated for experimentally produced IC (PIV-anti-PIV) and then applied to the analysis of IC in human sera. In the case of a renal transplant patient with CMV viremia whose free Ab titers were less than 100, the method unambiguously permitted the IC bound anti-CMV titers to be determined.

The importance of nuclear magnetic relaxation dispersion (NMRD) profiles in MRI contrast media development.

Observation of the relaxivity of MRI contrast media over a wide range of magnetic fields is not only necessary for predicting their efficiency at any field but also compulsory for understanding and improving their mechanisms of action. The best experimental approach to this problem is the field cycling method, which allows the exploration of nuclear relaxation over a broad interval of magnetic field intensity but requires a specially dedicated instrument called a relaxometer. Particularly relevant are the relaxivity profiles of the two chelates Gd-DOTA and Gd-DTPA.

Protein-bound polyamines in the plasma of mice grafted with the Lewis lung carcinoma.

Protein-bound polyamines were isolated from the plasma of mice using antipolyamine antibodies covalently linked to magnetic latex spheres. Their subsequent separation by polyacrylamide gel electrophoresis (PAGE) showed that in plasma from normal mice, 3 proteins (27, 55 and 82 kDa) carrying polyamines could be visualized, whereas in mice bearing the Lewis lung carcinoma at least 8 other proteins of higher molecular mass (5 of 94, 110, 130, 145 and 160 kDa, and 3 of greater than 170 kDa) had bound polyamines. These protein-bound polyamines could be detected from the first week after tumour graft; they increased during the second and third week but decreased thereafter.

[Demonstration of polyamines bound to plasma proteins during tumor growth].

Using antipolyamine antibodies covalently bound to magnetic latex spheres, we have been able to isolate from the plasma of mice bearing the Lewis lung carcinoma, 8 proteins with bound polyamines. These proteins are not found in the plasma of healthy mice or in those suffering with chronic inflammatory infection.

Transglutaminase activity and putrescine-binding capacity in cloned cell lines with different metastatic potential.

An inverse correlation was found between cellular transglutaminase activity and metastatic potential of four cloned cell lines derived from a primary nickel-induced rat rhabdomyosarcoma. Cellular transglutaminase activity as assessed with endogenous cellular protein or exogenous methylated casein was greatest in the clone F9-4/8 which is the least metastasizing. When the putrescine-binding capacity of one cellular derived protein - fibronectin - was examined with exogenous transglutaminase, it was found that the fibronectin derived from the clone F9-4/8 showed the lowest binding capacity compared with those from the other clones.

Red blood cell polyamines in mice bearing the Lewis lung carcinoma (3LL) and in patients with bronchopulmonary cancers.

Experimentally, during Lewis lung carcinoma (3LL) growth, the red blood cell (RBC) polyamine levels increase with tumor volume and are inversely correlated to tumoral concentrations of spermidine and spermine. The RBC level of spermidine is continually correlated to both the volume and the tumoral concentration of this polyamine. Clinically, high levels of RBC polyamine are observed in cases of squamous-cell carcinoma or anaplastic cancer, and not in cases of adenocarcinoma.

A quantitative and qualitative study of the transglutaminase-mediated insertion of polyamines into plasma proteins from patients with bronchopulmonary cancer.

The transglutaminase-mediated incorporation of putrescine into proteins of plasma derived from 44 patients with histologically defined bronchopulmonary cancer was compared with that from an age-matched group of 18 patients hospitalized with non-malignant diseases. Two types of kinetic data were obtained over a range of putrescine concentrations 0.09 mM to 0.

The competitive inhibition of tissue transglutaminase by alpha-difluoromethylornithine.

The transglutaminase-mediated insertion of putrescine into casein was inhibited competitively by alpha-difluoromethylornithine (alpha-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Preincubation of the amine acceptor (casein) or the enzyme itself with the inhibitor did not affect enzyme activity. Alpha-DFMO is a poorer substrate for transglutaminase (Km = 2.

Differences in polyamine availability and insertion into fibronectins released from normal and transformed cells.

1. Fibronectin released from transformed rat kidney cells compared with that released from normal rat kidney cells shows a 50% increase in amino group availability. 2.

[Immunochemical evidence for the specific binding of putrescine to plasma proteins including fibronectin].

Human plasma contains proteins capable of binding 14C putrescine by the action of Ca++ activated transglutaminase. These proteins have molecular weights from 32 to 220 K and above. One of these (with a molecular weight of 220 K) has been identified as fibronectin by the use of an antifibronectin antiserum.

An automatic continuous flow method for the determination of antipolyamine antibodies in human sera.

Latex particles with covalently bound polyamines were used to detect antipolyamine antibodies in human sera in manual and automated nephelometric assays. There was good correlation between the two assays though in the former aggregate size was measured after 24 h incubation whereas in the latter monomer loss was determined after 1 h.

Evidence for natural antibodies (IgG) to polyamines in human sera.

Human sera contain IgGs that react with latex-putrescine spheres. Identification of the IgGs has been achieved by polyacrylamide gel electrophoresis and their reaction with peroxidase-labeled antihuman gamma-chain-specific antibodies. The inhibition of IgG fixation to latex-putrescine spheres by free spermine and putrescine provides evidence that these IgGs are specific for polyamines.

The preparation of latex particles with covalently bound polyamines, IgG and measles agglutinins and their use in visual agglutination tests.

Carboxylated latex particles were substituted with side arms terminating in primary amine and hydrazine groups. The particles were coupled to aldehyde groups generated on glycoproteins which were treated with sodium periodate. Particles having the alipathic primary amine putrescine hapten as the sole substituent and particles linked to glycoproteins such as measles agglutinins and IgG were used to detect the presence of the corresponding antibodies or antigens in biological fluids by agglutination tests.

Nephelometric assay of antigens and antibodies with latex particles.

Antigens and antibodies covalently bound to latex particles have been used to detect specifically corresponding antibodies and antigens by nephelometry. A systematic study of factors such as wavelength, angle of observation, concentration of latex particles, and reaction time has permitted us to develop a procedure suitable for routine estimation of antigens and antibodies in the nano and picogramme range.