Specificity of peroxidase-labeled antibodies to rabbit, mouse and human immunoglobulins

The comparative study of different types of conjugates (antirabbit, antimouse and antihuman) has shown that gamma globulin fractions actively interact not only with homologous peroxidase-labelled antibodies, but also with heterologous ones. Cross reactions were most pronounced between antimouse conjugates and the preparations of human gamma globulin and, vice versa, between antihuman conjugate and mouse gamma globulin. The study has shown that the main cause of cross reactions is the presence of common antigenic determinants in the preparations of mouse and human gamma globulin.     

Evaluation of the prophylactic effect of gamma globulin in meningococcal infection.

Results of two controlled epidemiological tests evaluating the prophylactic effect of gamma globulin of Monogolian and Soviet production against meningococcal infection are presented. Observations were carried out on children aged 3 months to 4 years, not attending children's establishments. The results of the observation revealed the following prophylactic effect of gamma globulin of Mongolian production in the first two months after administration: index of efficiency--5.0, coefficient of efficiency--80%, P greater than 0.01. The efficiency of the prophylactic effect of Soviet gamma globulin was limited to one month: the index of efficiency amounted to 5.3, the coefficient of efficiency to 82.2%, P greater than 0.01. The course of meningococcal infection in the children who had received gamma globulin was less severe than in the children of the control group. Lethal outcome was recorded only in the group of children who had not received gamma globulin. The duration of the prophylactic effect of gamma globulin was found to depend on the height of the titres of specific antibodies in the preparation. The preparations are recommended as prophylactic means for children aged from 3 months to 4 years in doses of 1.5 ml (one dose) in the foci of meningococcal infection.     

Development of a method of obtaining peroxidase conjugates with anti-immunoglobulin for immunoenzyme analysis

The results of the successive stages of preparing peroxidase-labeled anti-immunoglobulins are presented. The schedules for the immunization of animals have been worked out with a view to prepare antisera to human gamma globulin and, subsequently, to isolate antibodies from them by different methods. The preparations obtained with the use of immunosorbent have proved to possess the highest serological activity. As the result of these investigations, a new method of binding, a modification of the glutaraldehyde method, has been proposed. The conjugates obtained by the proposed method are characterized by high serological activity, stability and good reproducibility.     

Effect of anti-sperm gamma globulins on metabolism of human spermatozoa.

Rabbits were immunized with saline extracts of human spermatozoa Presence of antibodies to spermatozoa was confirmed by Ouchterlony gel diffusion, microscopic sperm agglutination, and immunofluorescent techniques. Anti-sperm gamma globulin significantly decreased the average oxygen consumption of 10⁸ washed human spermatozoa. Sperm incubated in the presence of anti-sperm gamma globulin had a significantly lower rate of lactic acid accumulation than sperm incubated in normal rabbit gamma globulin. It appears that anti-sperm antibodies can influence both aerobic respiration and glycolysis of washed human spermatozoa.     

The effect of human gamma globulin on tbe production of antibodies to the surface antigen of the hepatitis B virus in an experiment on animals

The biological assay for the determination of HBsAg in gamma globulin is proposed. The assay is based on the immunizing action of immune complexes and some specific features of secondary immune response. For this purpose, the highly purified preparation of HBsAg is injected into guinea pigs. Subsequently the animals receive human gamma globulin. An increase in the titer of anti-HBsAg antibodies in the animals preimmunized with the antigen is indicative of the presence HBsAg in human gamma globulin. In accordance with another variant of this assay, human gamma globulin is introduced into mice and 21 days later the animals are immunized with HBsAg. The appearance of anti-HBsAg antibodies in the animals, previously treated with gamma globulin, on day 4 after the injection of the antigen indicate that the animals experience the second penetration of HBsAg, i. e. its presence in previously injected human gamma globulin.     

Labeling of human IgG with rhodium-105 using a new pentadentate bifunctional ligand

We report the labeling of human gamma globulin with the 105Rh complex of a new pentadentate bifunctional ligand, 1,7-bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine. Complexes of this ligand with 105Rh were prepared by refluxing rhodium carrier spiked with 105Rh at pH9 in bicarbonate buffer. The complex was treated with an excess concentration of thiophosgene to prepare the isothiocyanate derivative which was extracted into CHCl3. The CHCl3 extract was dried and dissolved in DMF and reacted with a borate solution of human gamma globulin. Labeling yields were generally high and varied from 73% to 93%, depending upon the concentration of human gamma globulin and the isothiocyanate derivative of the complex used. The overall recovery of rhodium activity varied from 59% to 75% without taking into account activity lost due to decay. The conjugation reaction was complete by 4 h. From 0.4 to 8.5 atoms of Rh could be incorporated per molecule of protein by this method. The activated isothiocyanate complex did not show any degradation when stored at room temperature for up to 4 days and then used for conjugation.     

Development and trial of a heterogenous antistaphylococcal gamma-globulin

Antitoxic and antibacterial equine gamma globulin for the treatment of staphylococcal infection has been developed and introduced into medical practice. The pronounced effectiveness of the preparation has been demonstrated in comparison with the ineffectiveness of traditional therapeutic measures. The development of mild forms of the serum disease in 33% of cases indicates that further investigations on the purification of gamma globulin preparations are necessary.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Sex differences in the subunits of glutathione-S-transferase isoenzyme from rat and human kidney

Glutathione-S-transferase (GST) isoenzymes were purified from cytosolic preparations from kidneys of male and female rats and kidney cortical specimens from 2 male and 1 female human subjects. GST isoenzyme expression was analyzed by SDS-PAGE, measurement of catalytic activities with specific substrates and determination of their subunits by ELISA and Western blotting using specific antibodies. GST from female rat kidneys showed a preponderance of subunits 3 and 4; levels of these isoenzymes were 3-4 times greater in females than in males. Levels of subunits 1 and 2 were 1.5-2 times greater in the male rat kidneys. Additional minor bands at 24 and 22 kD were observed in GST preparations from both male and female rat kidneys while a band at 25.3 kD was observed only in the male rat kidney. These bands did not react with antibodies to GST 1-1, GST 2-2 or GST 3-4. Both male and female human kidney samples contained GST isoenzymes comparable to the near-neutral (25-5 kD) and basic forms (25 kD) of GSTs found in human liver. In addition a 28-kD band was present in GST preparations from both male and female human kidneys. Additional bands at 29 and 25.2 kD were present only in male human kidneys. Both the kidney cytosol and the total GSTs prepared from female rats shared 2- to 4-fold greater activity with 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one than those from males. The measurement of specific subunit amounts by ELISA were in agreement with these results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Locomotion and adhesion of neutrophil granulocytes : Effects of albumin, fibrinogen and gamma globulins studied by reflection contrast microscopy

Proteins as well as materials of low molecular weight have marked effects on the rate of locomotion, adhesion and cell shape of human neutrophil granulocytes in vitro. Plasma protein preparations differ qualitatively with respect to their chemokinetic activity. Human serum albumin (HSA), fibrinogen and acid-treated gamma globulin without polymers have a positive chemokinetic effect on neutrophils suspended in Gey's solution. Standard gamma globulin (SGG) or acid-treated gamma globulin with polymers have marked negative chemokinetic activity. Three different mechanisms are presumably responsible for the low rate of locomotion observed in Gey's solution alone, Gey's solution containing acid-treated gamma globulin with polymers or SGG, respectively: (a) too firm adhesion to the substratum; (b) lack of adhesion to the substratum; and (c) impaired capacity to perform shape changes. The relationship between attachment of cells to the substratum and the rate of neutrophil locomotion has been investigated. It appears that the pattern of adhesion rather than cell attachment as measured by the proportion of neutrophils adhering to the substratum is a meaningful correlate to locomotion. Two different patterns of adhesion can be distinguished by means of reflection-contrast microscopy: (a) the pattern characterized by uniform grey areas is compatible with efficient locomotion; (b) a pattern characterized by large black areas at the cell periphery. It is associated with neutrophils in Gey's solution which fail to displace themselves efficiently. This suggests that reflection-contrast microscopy may be helpful in distinguishing contacts allowing locomotion to occur from contacts impeding neutrophil locomotion.     

Recombinant gamma interferon enhances natural killer cell activity similar to natural gamma interferon

The enhancement of human natural killing activity by recombinant human gamma interferon (IFNγ) and natural human IFNγ were similar over a wide concentration range. Enhancement of natural killing activity by both interferons was neutralizable by antibody to natural IFNγ, as well as by antibody to a synthetic peptide representing the first 20 N-terminal amino acids of IFNγ provide conclusive evidence that IFNγ is responsible for the enhanced natural killing activity seen in IFNγ preparations.     

Antitoxin in human pertussis immune globulins

The level of antitoxin i.e. neutralizing antibodies to pertussis toxin, or lymphocytosis promoting factor, was determined in six pertussis immune globulin preparations from different manufactures. A comparison with antitoxin levels after natural pertussis disease in adults showed that pertussis immune globulins did not contain more antitoxin than convalescent phase sera, i.e. they had very low antitoxin content for specific immune globulins. Agglutinin and anti-FHA titres were relatively higher in immune globulins, probably reflecting a difference between the antibody response elicited by whole cell vaccines used for hyperimmunization in immune globulin production and by natural disease. The low antitoxin content of currently available pertussis immune globulin preparations could explain the inefficacy or conflicting results obtained with these products in prophylaxis and therapy of whooping cough.     

Determination of antibodies to mycobacterial antigens in tuberculosis by erythrocyte immunoadsorption with a rosette marker

A solid-phase enzyme immunoassay system for the determination of antibodies to mycobacterial antigens, based on the method of erythrocyte immunoadsorption in microchambers for immunological reactions, has been developed. To detect antibodies specifically bound with the solid-phase antigen, the affinity rosettes of Staphylococcus aureus strain Cowan I, carrying protein A, with erythrocytes conjugated with human gamma globulin have been used. The significant correlation of the titers of 34 sera, determined by means of erythrocyte immunoadsorption, with extinction values obtained in the solid-phase enzyme immunoassay of antibodies to Mycobacterium tuberculosis has been established. The coincidence of the results in 92% of cases has been noted.     

Studies of Fc gamma receptors of human B lymphocytes: phospholipase A2 activity of Fc gamma receptors

The presence of phospholipase A2 activity within human B cell Fc gamma receptors was investigated. Lysate produced by detergent treatment of chronic lymphocytic leukemia cells that had 1% of the cells surface radioiodinated was subjected to affinity chromatography by using either rac-1-(9-carboxynonyl)-2-hexadecylglycero-3-phosphorylcholine-Sepharose (PC-Sepharose) or heat-aggregated human IgG-Sepharose 4B conjugate (IgG-Sepharose). The materials eluted from both adsorbants by ethylenediaminetetraacetate- or urea-containing buffer were further purified by gel filtration and isoelectric focusing in the presence of 6 M urea. Both isolated PC- and IgG-binding materials were homogeneous, when judged by gel filtration and isoelectric focusing, and had identical isoelectric points (pI = 6.5), peptide maps, and amino acid compositions. Furthermore, both preparations catalyzed equally the hydrolysis of phosphatidylcholine to release fatty acid from the 2 position. Optimal enzymatic activity depended on the presence of Ca2+, was maximal at pH 9.5, and was augmented by Fc gamma fragments. Both preparations specifically bound to the Fc portion of IgG and inhibited human antibody-coated erythrocyte rosette formation by peripheral mononuclear cells. Our data thus demonstrate the identity of PC- and IgG-binding materials and suggest that a functional activity of the human B cell Fc gamma receptor is the generation of phospholipase A2 activity within the plasma membrane.     

Maintenance of plasma-derived proteins at much lower concentrations in the uterine lumen of the rabbit: a kinetic study of passage.

Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.     

Development of a Soviet immunoenzyme test system for the quantitative determination of human immunoglobulin E

An EIA system for the quantitative determination of human IgE was developed. As the solid phase, polystyrene microplates sensitized with the gamma globulin fraction of sheep antiserum to human IgE were used in this system. Peroxidase conjugate with IgE was prepared with the use of periodate technique. The optimum parameters for the sorption of antibodies on polystyrene were determined: protein concentration, the pH of the buffer, temperature, the time of fixation, the working dilution of the conjugate. As the substrate of peroxidase activity, ortho-phenilenediamine solution with hydrogen peroxide was used. The result was evaluated by means of a photometer, model Titertek Multiskan MC (Flow Laboratories Ltd., Great Britain), at the wavelength 429 nm. The new EIA system proved to be specific and detected IgE in the sera under investigation at a concentration of 10(-9) g/ml. The system permitted the determination of the normal level of IgE in a group of healthy adults, this level being, on the average, 76 +/- 9 kU/1.     

Modification of the immune reaction by antigen-immunosuppressive agent-conjugates. III. Physiocochemical, antigenic and functional properties of 6-mercaptopurine coupled carrier proteins]

6-mercaptopurine (MP) conjugates of bovine gamma globulin (BGG) and human serum albumin (HSA) with increasing numbers of MP-groups per protein molecule were prepared and some physicochemical and antigenic properties studied. The electrophoretic mobility increased proportionally to the degree of substitution. In the same manner the formation of high molecular weight aggregates increased. A loss of BGG and HSA specific determinants was observed too. The physicochemical and antigenic properties of these antigen-suppressive agent-conjugates with low and medium coupling degree (to MP26-BGG and MP20-HSA) were similar to the unmodified antigens. The affinity of rabbit anti-DNP-antibodies decreased from K0 = 1,4 - 10(6) M-1 of the unmodified antibodies to 6,5 - 10(5), 3,6 - 10(5) and 3,4 - 10(5) M-1 of the antibodies conjugated with 15, 21 and 29 MP-groups per antibody molecule. The precipitation capacity of the MP-conjugated antibodies decreased to 89, 85 and 61% for the same coupling ratios. These results suggest that the upper limit of the coupling ratio of the antibodies is 20 to 1.     

Effect of multiple gamma globulin administration on the effectiveness of seroprophylaxis in hepatitis A

The data provided by immunological and epidemiological studies carried out to determine the influence of the multiple injections of 10% commercial gamma globulin on the level of antigamma globulin formation and, in this connection, on the effectiveness of the prophylaxis of hepatitis A are discussed. A sharp increase in the titers of antigamma globulin in persons receiving multiple gamma globulin injections is shown. The increased amount of antibodies to gamma globulin, appearing as the result of the multiple use of this preparation, decreases the effectiveness of the seroprophylaxis of hepatitis A.     

Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats.

Initial studies of the prophylactic effect of parenterally administered respiratory syncytial virus (RSV)-neutralizing antibodies in cotton rats indicated that virus replication in lung tissues was restricted when animals with preexisting antibody titers in serum of 1:100 or more (as measured by plaque reduction) were challenged intranasally with 10(4) PFU of virus. Subsequently, a therapeutic effect of parenterally administered RSV antibodies (present in human gamma globulin) was demonstrated in both cotton rats and owl monkeys. Parenteral inoculation of RSV-infected cotton rats or owl monkeys with purified human immunoglobulin licensed for intravenous administration in humans (IVIG) effected a 10(-1.7) to 10(-2.7) reduction in the level of pulmonary virus at the height of infection. Because of these encouraging results, we examined topical administration of IVIG to determine whether it was also effective and whether it offered an advantage over the parenteral route with regard to simplicity and the dose required for full therapeutic effect. IVIG (0.025 g/kg) administered topically by the intranasal route to anesthetized cotton rats at the height of RSV infection effected a 10(2.2)-fold reduction in viral titers of pulmonary tissues and a complete clearance of detectable virus in 92% of the animals within 24 h. In contrast, 4 g of IVIG per kg was required to produce a comparable therapeutic effect when the material was administered parenterally. Thus, the therapeutic effect of IVIG was 160 times greater by the topical route than by parenteral inoculation.     

Effectiveness of the seroprophylaxis of tick-borne encephalitis depending on the titer of homologous gamma-globulin antihemagglutinins

To give a rationale for using homologous gamma globulin with antihemagglutinin titers of 1 : 20 to 1 : 80 for the prophylaxis of tick-borne encephalitis, 5-year observations covering all persons attacked by ticks in one of the intensive natural foci of the disease in the Western Urals have been made. The threefold statistically significant difference in the morbidity rate of tick-borne encephalitis between groups of persons immunized and not immunized with gamma globulin has been shown.