Ability of FAB-fragments of normal rabbit and human gamma globulins to suppress human lymphocyte blast transformation induced by phytomitogens]

Monovalent and bivalent Fab-fragments of normal human or rabbit gamma-globulin suppressed blasttransformation of human lymphocytes induced by phytohemagglutinin and concanavalin A. Peptic F(ab)2-fragments from highly-purified rabbit anti-DNP antibody displayed suppressing activity similar to that of the fragments of normal gamma-globulin. Fab-fragments affected blasttransformation when added to lymphocytes either simultaneously with the PHA or 24 and 48h after the mitogen. The data obtained may indicate that the inhibiting of lymphocyte blasttransformation produced by the gamma-globulin fragments was not caused by their competing with mitogens for the receptors on the target-cell; the Fab-fragment activity was probably determined by the structures located outside the antibody active centre.     

Fluorescent FAB-fragments of antibodies. Comparative assessment and utilization in immunofluorescent analysis]

The authors present the results of a comparative study of the immunofluorescent activity and the specificity of fluorescent Fab-fragments of antibodies to R. prowazeki, D. sibericus, and B. pertussis obtained by the enzymatic hydrolysis of specific immunoglobulins with papain. Fluorescent Fab-fragments of antibodies possessed the same sensitivity and specificity as the homologous fluorescent antibodies, but had an advantage of a much weaker capacity to nonspecific fluorescence and a relatively higher staining properties. Fluorescent Fab-fragments of antibodies, in contrast to the fluorescent antibodies of the same specificity, permitted to detect the antibody searched in the concentrated crude material, and thus increased the possibilities of the immunofluorescent analysis.     

FAB immunoglobulin fragments. I. The comparative characteristics of the serological and virus-neutralizing properties of a gamma globulin against tick-borne encephalitis and of the FAB fragments isolated from it]

A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a marked virus-neutralizing activity. The mean value of a logarithm of the neutralization index was 2.65 +/- 0.2 for Fab-fragments and 3.74 +/- 0.38 for gamma-globulin (P less than 0.01).     

Dependence of the specific activity of antitetanus immunoglobulin on the degree of its fragmentation

Antitetanus immunoglobulin preparations with the increasing content of Fab-fragments (15, 30, 53%) have been obtained under specific experimental conditions. Tests for specific activity have revealed an insignificant decrease (13%) in this activity in the preparation containing 15% of Fab-fragments and its sharp drop in the preparations containing 30-50% of Fab-fragments. The specific activity of antitetanus immunoglobulin has been found to be related to the degree of its fragmentation.     

Dependence of the specific activity of an immunoglobulin against tick-borne encephalitis on the degree of its fragmentation

Immunoglobulin preparations against tick-borne encephalitis with the increasing content of Fab-fragments (from 16% to 45%) have been experimentally obtained. As revealed by testing these samples in vivo for specific activity (in the biological neutralization test), the preparations containing 16% of Fab-fragments show no perceptible decrease of specific activity; its sharp decrease (2-16 times) can be observed in preparations with a high degree of fragmentation (the content of Fab-fragments being equal to 30-45%).     

Synthesis of lipid A analogs. Obtaining conjugates of 6-phosphate 2-desoxy-2-(3-hydroxymyristoyl)amino-D-glucose with polymer carriers and their immunological properties

The derivatives of 2-acylamino-6-O-(2-aminoethyl) phosphono-3-deoxy-D-glucose acylated with acetic or D,L-3-hydroxytetradecanoic acid were obtained, and their 31P-and 13C-NMR spectra investigated. These haptens were bound with a polysaccharide (Ficoll) or proteins (albumins, bovine gamma-globulin). The protein conjugates were immunogenic in rabbits, specific antibodies against the hapten being revealed by two immunochemical methods. As shown by the enzyme-linked immunoadsorbent assay, the specific rabbit antiserum reacted with lipid A from Yersinia pseudotuberculosis.     

Fatty acid acylated Fab-fragments of antibodies to neurospecific proteins as carriers for neuroleptic targeted delivery in brain.

A method for targeted delivery of neuroleptics from blood in brain based on using Fab-fragments of antibodies to antigens of brain glia cells (acid gliofibrillar antigen and alpha 2-glycoprotein) is suggested. The essence of the technique is that the molecule of neuroleptic (trifluoperazine) is conjugated with Fab-fragments of these antibodies. The conjugate thus obtained is modified by stearoylchloride in the system of Aerosol OT reversed micelles in octane. The study of the distribution of 125I-labelled conjugates in the rat organism after intracordial introduction is performed. On the contrary to the nonmodified conjugates and conjugate, containing fatty acylated Fab-fragments of antibodies, nonspecific to the rat brain, the conjugate of trifluoperazine with stearoylated Fab-fragments of antibodies to neurospecific antigens accumulate in brain tissues. The drastic increase of the neuroleptic activity of trifluoperazine resulting from its coupling with stearoylated Fab-fragments of antiglial antibodies is observed.     

Therapeutic effectiveness of gamma-globulin on experimental burns

Experiments on rabbits were made to examine excretory-absorption liver function and the blood kallikrein-kinin system after thermal burn and treatment with gamma-globulin immune to burn skin toxin. Immunotherapy leads to a more rapid and effective recovery of the functional indicators in the burnt animals than in burns without or with the treatment by nonimmune preparations. It was demonstrated that specific detoxification plays an important role in multiple modality treatment of burn disease.     

Twenty-four hour changes in lysozyme levels, total plasma protein concentration, gamma-globulin concentration, white blood cell count and numbers of lymphocytes and granulocytes in the peripheral blood of chinchillas Chinchilla laniger M. and rabbits Oryctolagus cuniculus L

Twenty-four hour changes in lysozyme level, total plasma protein concentration, gamma-globulin concentration, leucocyte number and different forms of leucocyte in the peripheral blood of male chinchillas and rabbits were investigated. All investigated parameters in both species showed statistically significant 24-hr changes, but only total plasma protein concentration in rabbits and gamma-globulin concentration and leucocyte number in chinchillas have a circadian rhythm character. The cosine curve characteristics for these rhythms are as follows; acrophases 0.45 + 2.18 hr, 10.02 + 4.29 hr, 2.14 + 3.20 hr; amplitudes 0.48 + 0.32 g%, 0.34 + 0.41 g%, 1.12 + 0.88 X 10(3)/min3; mesors 6.10 + 0.22 g%, 2.23 + 0.21 g%, 8.78 + 0.65 X 10(3)/mm3, respectively.     

Isolation, crystallization, and preliminary x-ray study of a Fab fragment of monoclonal antibody against human interleukin-2 and its complex with antigenic peptide]

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.     

Immunologic effects of morphine administration in rabbits.

Long-term effects of morphine administration or immunologic test responses were studied in female rabbits. Implantation of morphine-containing pellets was found to be more effective than injection of morphine sulfate solutions in promoting increased serum binding of 140-morphine. A large part of the increased morphine binding by sera associated with administration of morphine was found in serum fractions containing gamma-globulin and was absent in gamma-globulin-free fractions. These sera showed some degree of specificity for the morphine configuration in tests with other narcotics. They also gave positive immunologic test reactions in passive hemagglutination and radial immunodiffusion tests involving serum albumins conjugated with morphine derivatives. Other evidence for immunologic responsiveness against morphine by morphine-pretreated rabbits was shown by cutaneous hypersensitivity reactions against morphine-carrier conjugates and by a diminution of the serum morphine-binding response in rabbits given an immunosuppressive dose of cyclophosphamide. Failure of naloxone, a morphine antagonist, to alter the serum morphine-binding response suggested that serum levels of the morphine-binding globulin studied here were not direclty related to morphine withdrawal.     

Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody.

Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named 'Fab-lipids' in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensional pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were qualified using micro-fluorimetry.     

Large-scale purification and preliminary structural studies of MAC 182, a gibberellin-binding monoclonal antibody.

The large-scale purification of the anti-gibberellin monoclonal antibody, MAC 182, is described. N-Terminal amino acid sequences of the heavy and light chains were determined and compared with those of known antibodies. Fab-fragments were prepared and purified to a state suitable for crystallization.     

Antibodies to meningococcus polysaccharides in nonspecific gamma-globulins from different regions of the world

Seventy-one batches of nonspecific gamma-globulin obtained from France, USSR and Mongolia were studied for presence of specific antibody to group A and C meningococcus polysaccharide. Specific activity was tested by two methods: radioimmunoassay (Lyon) and reaction of passive haemagglutination inhibition (Moscow). Antibodies were detected in all the gamma-globulin batches tested, in some of them at high titres. The summary results indicated that approximately equal levels of specific A antibodies were present in preparations obtained from the different regions of the world. Antibodies to group C polysaccharide showed considerable variation in level from selection country to country; the highest level of C antibodies was in gamma-globulin from France. The authors feel entitled by the results to recommend testing of nonspecific gamma-globulin, selection of batches with a high level of specific antimeningococcus antibodies, and their judicious use.     

Method of isolating and controlling fluorescent Fab fragments of antibodies against horse serum proteins

For the first time the lyophilized fluorescent Fab-fragments of rabbit antibodies to horse serum protein, suitable for detecting different antigens and antibodies to these antigens, have been obtained by the specially developed method. The criteria to be used in the control of the antispecific fluorescent fragments of antibodies have been described and the methods of their control before and after lyophilization have been developed. The use of the antispecific fluorescent Fab-fragments of antibodies has been shown to considerably accelerate and simplify the indirect immunofluorescent assay.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

SERUM ‘ANTI-MYELIN ANTIBODIES’: A SPECTROFLUOROMETRIC ASSAY PROCEDURE1

A simple spectrofluorometric procedure has been devised to determine serum antibodies, directed to constituents of the myelin sheath. It is an adaptation of the indirect immunofluorescent technique. A suspension of highly purified bovine myelin is incubated successively with a test rabbit serum and fluoresceinisothiocyanate-conjugated anti-rabbit gamma-globulin. Intensity of fluorescence in the final myelin suspension is determined spectrofluorometrically. Sera from rabbits with experimental allergic encephalomyelitis, induced by whole bovine spinal cord, generally gave fluorescence at least 10 times that of normal rabbit serum. Fluorescence of sera with high demyelinating activity was more intense than that of sera with equivocal demyelinating activity. The assay is specific for immunoglobulins directed to myelin constituents, organ-specific and species-independent. Rabbit anti-galactosylceramide serum with known demyelinating activity gave high fluorescence similar to that in sera of rabbits inoculated with whole spinal cord. Galactosylceramide could absorb a substantial portion of‘anti-myelin antibodies’of the anti-galactosylceramide serum but it did not absorb‘anti-myelin antibodies’of serum of rabbits with whole tissue-induced experimental allergic encephalomyelitis. This assay system may be useful for further studies of ‘anti-myelin antibodies’.     

Effect of gamma globulin on reactivity of the organism. V. Types of biological activity of gamma globulin preparations]

Biological activity of 110 series of commercial gamma-globulin preparations was studied; they were found to contain placental antigens, group-specific blood substances, gonadotropic hormones and antibodies to them. Placental antigens were found in 12% of placental and abortive gamma-globulin batches in titres of 1 : 2--1 : 16; no placental protein was revealed in donor gamma-globulin. There were group-specific blood substances in all the batches of placental and abortive gamma-globulin studied (in titres of 1 : 138--A, 1 : 112 B in the placental gamma-globulin and in titres of 1 : 48.9--A, 1 : 32--B in the abortive gamma-globulin). In the preparations from the venous blood group-specific substances were either absent or present in lowe titres only (1 : 2). The value of gonadotropic hormones in the placental gamma-globulin batches constituted 873+/-157, and in the abortive--991.4+/-147 IU/l; no gonadotropins were revealed in donor gamma-globulin. The mean titres of antibodies to gonadotropin hormone in the gamma-globulin preparations made of placental blood constituted 1 : 236+/-32, of abortive--1 : 131+/-16.6, and of the venous blood--1 : 46+/-24.7. The presence of biologically-active substances in the gamma-globulin preparations pointed to the necessity of increased requirement of their quality; additional requirements to its standardization proved to be also necessary.     

Effect of immunization with small doses of antigen on the development of experimental atherosclerosis

Immunization of rabbits suffering from experimental hyperlipemia with human gamma-globulin (a total dose of 150 mg) or with disintegrated yeast Candida albicans (a total dose of 62 mg) inhibited the development of hyperlipemia and atherosclerosis. The effect was more pronounced in animals immunized on the 6th experimental week than in those immunized on the 9th week.     

The different electrophoretic forms of post gamma-globulin their antigenic identity and their structural variability.

Post gamma-globulin, first described as a constant component of protein of cerebrospinal fluid and urine from patients with tubular disorders has also been found in other biological fluids. Post gamma-globulin from a single individual always migrated as several bands after storage. Three electrophoretic forms, immunochemically identical, have been isolated by gel chromatography, preparative continuous flow electrophoresis and ion-exchange chromatography. Their molecular weight was found to be approximately between 11 000 to 12 000. No difference between the three forms could be detected. The N-terminal amino acids were found to be Lys, Arg and Leu respectively for the three forms of post gamma-globulin. The "slow" and "fast" forms of post gamma-globulin seemed to differ by elimination of small basic peptides or amino acids from the N-terminal end of the protein. No enzymatic activity of post gamma-globulin was found, but this requires further investigations.