Quantification of antigens with haemolysing antibodies exemplified by the bovine J blood group system*

• 1 The J blood group activity of red cells is measured in terms of 50 % haemolysis (‘direct test’), that of dissolved or suspended samples in terms of 50 % haemolysis inhibition (‘indirect test’) in a standardized bovine J system.
• 2 The volume of J-containing sample required for a 50% haemolysis inhibition decreases with increasing J activity.
• 3 The volume of anti-J required for a 50% haemolysis of J-positive erythrocytes also decreases with increasing J activity.
• 4 The use of antigen units (U_Ag) was introduced to serve as a measure of J activity of dissolved or suspended samples.
• 5 Antigen units were also used to characterize J-containing red cells. This was made possible by measuring the relation of the direct test (on red cells). Thus, a relatively simple method of determination of red cell U_Ag is obtained.
• 6 It was confirmed by absorption experiments that erythrocytes containing high concentrations of antigen require relatively low amounts of antibody to bring about a 50 % haemolysis, but are able to bind a relatively high excess of antibody.

     

Two types of haemolytic activity of detergents

The nonionic detergent Triton X-100 at concentrations of about 0.003 to 0.008% causes swelling followed by the haemolysis of erythrocytes suspended in 160 mM KCl. The rate of haemolysis increases with the increase in detergent concentration. Finally all the erythrocytes are haemolysed. The resistance of erythrocytes to this detergent decreases with an increase in temperature. The effect of Triton X-100 is explained by increased membrane permeability to KCl and colloid osmotic haemolysis. The anionic detergent, sodium dodecyl sulfate (SDS), at concentrations of about 0.003 to 0.001% causes the haemolysis of a certain number of erythrocytes. This number increases with an increase in detergent concentration. The resistance of erythrocytes to SDS increases with an increase in temperature. The effect of SDS is explained by direct disruption of membranes by the detergent.     

Feedback by early and late primary antisera on the primary and secondary adoptive immune responses of mice to burro erythrocytes

Experiments on antibody feedback inhibition of the immune response have confirmed that control is more effective against a primary response than against a secondary response. The cells producing antibodies in primary and secondary responses are different both in terms of number of IgFC and amount of antibody produced by individual IgFC (plaque size). Late primary anti-burro RBC sera (greater than 200 days), despite low titers, are, on a volume for volume basis, feedback inhibitors at least as good as early (8-12 days) primary antisera on primary responses but are more effective in suppressing secondary responses (B memory cells). Late primary antisera, due to the process of affinity maturation, have a high affinity for antigen. The suppressive effect of early and late antisera is equally removable by absorption with burro erythrocytes: a result which it is thought, decreases the likelihood of feedback by anti-idiotype being involved in the observed suppression. It is suggested that feedback antibody acts (a) in competition with receptors, inter alia removing antigen into immunologically irrelevant pathways, (b) by a process involving the linking of antigen to Fc receptors, and (c) as a blocking antibody coating B cells (Bm) or APC which are already binding epitopes, thus preventing their cooperation with specific helper or other accessory cells.     

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.     

Effect of adrenaline on glucose transport in red cells of Rana balcanica

The characteristics of 3-O-methyl-D-glucose (3-OMG) uptake by frog erythrocytes were studied. 3-OMG transport was increased by adrenaline. Although the transport is inhibited by phloretin, the lack of saturation kinetics suggests that a glucose transporter doesn't exist or that its affinity for glucose is extremely low. Frog Rana balcanica red cells suspended in an isotonic medium containing adrenaline enlarge rapidly to reach a new pH-dependent steady state volume. At pH 8.0, the cells swell less than at pH 7.3. This is explained by a differential pH effect on the two pathways controlling the movement of the cations: as pH becomes more acidic K+ loss decreases. On the contrary as pH becomes more acidic Na+ uptake increases. The increase in glucose transport after osmotic swelling and the inhibition of swelling-induced glucose transport by phloretin suggest that the glucose transport pathway in Rana balcanica erythrocytes may is a volume-activated channel.     

THE INFLUENCE OF DOSE FRACTIONATION ON RADIATION-INDUCED HAEMOLYSIS OF HUMAN ERYTHROCYTES

In this paper the radioresistance of human erythrocytes to radiation-induced haemolysis was studied by means of the split-dose technique. Human erythrocytes suspended in an isotonic Na-phosphatase buffer, pH7.4 were exposed under air to gamma radiation. Erythrocytes were irradiated with a single dose of 2.7kGy and with the same total dose split into two fractions with an interval between the subsequent exposures. It seems that postradiation processes initiated by the first radiation dose of definite magnitude can lead to an increase in the resistance of erythrocytes to haemolysis under the influence of subsequent dose. The maximal effect of the fractionation (50% decrease of haemolysis) was observed when the first fraction was equal to 0.3–0.4kGy and the interval between exposures was equal to 3h.     

Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Role of erythrocyte membrane lipids in the antagonism between vitamin A and D3

The "in vitro" lytic effect of retinol (35 microM) on various animal species erythrocytes is partially inhibited by cholecalcipherol. The inhibition observed is not related with reduction of retinol bound by erythrocytes membranes while it can be related with the amount of glycolipids not gangliosides of these cells. Instead, we have observed that the amount of retinol molecules bound to erythrocytes membranes and their haemolysis can be related with the gangliosides contents of red cells membranes.     

Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.     

Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure

Epsilon toxin (ETX) is a 33‐kDa pore‐forming toxin produced by type B and D strains of Clostridium perfringens. We previously found that ETX caused haemolysis of human red blood cells, but not of erythrocytes from other species. The cellular and molecular mechanisms of ETX‐mediated haemolysis are not well understood. Here, we investigated the effects of ETX on erythrocyte volume and the role of the putative myelin and lymphocyte (MAL) receptors in ETX‐mediated haemolysis. We observed that ETX initially decreased erythrocyte size, followed by a gradual increase in volume until lysis. Moreover, ETX triggered phosphatidylserine (PS) exposure and enhanced ceramide abundance in erythrocytes. Cell shrinkage, PS exposure and enhanced ceramide abundance were preceded by increases in intracellular Ca²⁺ concentration. Interestingly, lentivirus‐mediated RNA interference studies in the human erythroleukaemia cell line (HEL) cells confirmed that MAL contributes to ETX‐induced cytotoxicity. Additionally, ETX was shown to bind to MAL in vitro. The results of this study recommend that ETX‐mediated haemolysis is associated with MAL receptor activation in human erythrocytes. These data imply that interventions affecting local MAL‐mediated autocrine and paracrine signalling may prevent ETX‐mediated erythrocyte damage.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Estimation of relative antibody affinity at the level of the antibody-secreting cell during maturation of the immune response

The relative affinity of specific antibody secreted by mouse spleen cells following primary immunization with SRBC was estimated by competitive inhibition assay of antibody secreted by PFC as well as by inhibition of observed PFC number. Inhibition of direct and of indirect anti-SRBC plaque assays by the addition of specific antigen (SRBC stromata) gave sigmoid inhibition profiles from which the concentration of antigen required to inhibit 50% of the plaques (PI₅₀) was determined, Alternatively, the sum of the cube of individual plaque diameters (Σd³) provided a measure of total anti-SRBC antibody secreted by PFCs from which the concentration of antigen required to inhibit 50% of the antibody (Ab₅₀) was determined. Ab₅₀, rather than PI₅₀: (a) was a more sensitive measure of inhibition by antigen; (b) decreased following immunization indicating a progressive increase in mean antibody affinity; and (c) correlated with the results of hemolysin transfer experiments, an independent measure of mean affinity of circulating anti-SRBC antibody. From theoretical considerations, estimation of mean antibody affinity requires quantitative analysis of fractional antibody inhibition by antigen. Determination of Ab₅₀, rather than PI₅₀, provides an estimate of bound and of free antibody and therefore should provide a more valid estimate of the relative antibody affinity at the cellular level. Experimentally, utilizing Ab₅₀ analysis, the IgM and IgG responses of C3H mice to immunization with SRBC demonstrated a progressive increase in affinity during maturation of the immune response.     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes

We studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40–50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K-CI contransport which peaked 3 days after the last bleeding. The Na-K pump activity peaked at a similar time, but the % increase was twofold less than the K-CI contransport. There was very small increase in the activity of the Na-Li exchange, whereas the Na-H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K-Cl contransport and Na-K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80–90% of circulating cells). Transport rates were markedly increased (Na-K pump 9.6-fold, Na-H exchange 6.8-fold, Na-Li exchange 2.75-fold; K-CI contransport: 10–20-fold). When blood from PHZ-treated rabbits was incubated in vitro for 24–48 hour, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K-CI contransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 μM E-64, whereas 10 mM methylamine reduced the inactivation of the Na-H exchange and of the Na-Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K-CI contransport and inhibition of Na-K pump, Na-H exchange, and Na-Li exchange. These effects were abolished by DTT, with the exception of the Na-K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of cation transport in rabbit red cells. © 1993 Wiley-Liss, Inc.     

Penetration and entrapment of large particles in erythrocytes by electrical breakdown techniques

Human erythrocytes suspended in isotonic solutions were subjected to haemolysis by application of an electric field pulse to the cell suspension. The field strengths used were 12 and 16kV/cm, respectively; the pulse duration 40 microseconds. The lysed cells showed resealing properties. The permeability change of the membrane generated by the field pulse and by the subsequent osmotic processes were large enough to facilitate the penetration and entrapment of ferritin and Latex particles (diameter: 0.091 and 0.176 micron, respectively) as revealed by electron microscopy. Correct identification of the Latex particles in the electron-micrographs indicated that LOYTER et al. [J. Cell Biol. 66, 292 (1975)], who recently demonstrated the entrapment of Latex spheres in erythrocytes prepared by osmotic haemolysis mistook electron-dense bodies probably consisting of denaturated protein for Latex particles. Under conditions of osmotic haemolysis, carried out according to BODEMANN and PASSOW, particles could only occasionally be detected within the membrane itself and never within the cell interior, suggesting that the electrical haemolysis method is much more effective in the generation of large holes in the membrane.     

Erythrocyte antigen: isolation, biochemical and immunologic properties]

The authors suggest a simple method of obtaining erythrocytic antigen in considerable amounts. This antigen is of stromal origin, contains from 10 to 20% protein, and is relatively homogenous. With the concentration of from 1 to 50 microgram by protein the preparation represents a transparent solution; with greater concentrations the antigen is white, turbid, but is well dissolved and convenient for administration to the animals. In case of a single administration without any adjuvants the antigen is highly immunogenic in low doses by protein. To the optimal immunizing dose of erythrocytes (5 X 10(8)) correspond about 100 microgram of the antigen by protein. The primary response to the antigen is similar to the response to sheep red blood cells (SRBC). It is exceedingly effective for the formation of immunological memory. The level of secondary responses in the adoptive transfer to all the EAG doses always exceeded the secondary response to SRBC. By adding EAG into agar during the local hemolysis in gel test determined the avidity of the antibodies synthesized at various periods of the immune response to SRBC.     

Antigenic determinants of senescent antigen of human erythrocytes are located in sialylated carbohydrate chains of Band 3 glycoprotein.

Naturally occurring IgG autoantibody against Band 3 glycoprotein of human erythrocyte membrane (anti-Band 3) recognizes the erythrocytes modified with oxidizing or SH-blocking agents as well as senescent erythrocytes. Location of the antigenic determinants of Band 3 this autoantibody recognizes was investigated by competitive inhibition studies of the antibody binding to the modified cells. Autologous IgG binds to the modified erythrocytes, and purified Band 3 totally inhibits the binding. This inhibitory activity of Band 3 was not affected by digestion of Band 3 with various proteases. Treatment of Band 3 with endo-beta-galactosidase that destroys the poly-N-acetyllactosaminyl sugar chain of Band 3 or with neuraminidase resulted in loss of the inhibitory activity. Oligosaccharides released from Band 3 by hydrazinolysis effectively inhibited the binding of autologous IgG and 125I-labeled purified anti-Band 3 to the modified cells, whereas the oligosaccharides depleted of acidic components did not. Endo-beta-galactosidase and neuraminidase destroyed the activity of the oligosaccharides, but alpha-L-fucosidase did not. Furthermore, human lactoferrin that contains sialylated two N-acetyllactosaminyl units also exhibited potent inhibitory activity, and the activity was destroyed by endo-beta-galactosidase and neuraminidase. These results indicate that the antigenic determinants of Band 3 are located in sialylated poly-N-acetyllactosaminyl sugar chains. Based on this finding, mechanism of appearance of the antigen on senescent erythrocytes recognized by anti-Band 3 (senescent antigen) was discussed.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.