Heat Elution Research Articles

A New Reagent (ZZAP) Having Multiple Applications in Immunohematology

A reagent (ZZAP) containing a mixture of 0.1 M dithiothreitol (DTT) plus 0.1% cysteine-activated papain was found to dissociate IgG immunoglobulin from red blood cells (RBC) of patients having a positive direct antiglobulin test (DAT) although this could not be achieved with either chemical alone. In all 67 patients tested, ZZAP treatment of IgG sensitized RBC reduced the strength of the DAT, and in all 52 instances tested, this allowed for accurate Rh phenotyping using slide/rapid tube typing reagents. This included five examples in which spontaneous agglutination that occurred in saline or 6% albumin was eliminated by ZZAP. Thus, all red blood cell typing for Rh-Hr antigens was accomplished using slide/rapid tube reagents making unnecessary the use of saline reactive or chemically modified antisera. Kidd antigen typing is also possible after ZZAP treatment of IgG sensitized RBC. In regard to warm autoabsorption tests, ZZAP treatment of 14 RC samples having a positive DAT proved preferable to heat elution technics since equal or greater amounts of IgG were removed by ZZAP and little or no hemolysis resulted. ZZAP has no effect on ABH, Rh or Kidd antigens but denatures Duffy, MNSs and all Kell antigens tested (K:1-K:7, K:11-K:14, K:18, K:19). This should prove valuable in certain investigations of multiple alloantibodies and, moreover, may allow for better characterization of Kell antigens.

Elution of group-specific substance A from RBC of various subgroups of A and its effect on the agglutination of AX RBC.

Experiments are described which demonstrate that when the Landsteiner heat elution technique is used to prepare eluates from group A cells, some A substance as well as anti-A is released into the eluates. The concentration found is greatest from A1 RBC and least from Ax, suggesting a relationship with the number of antigen sites on RBC of the various subgroups. Further experiments have shown that the presence of this A-specific substance in eluates accounts for the well-know phenomenon that anti-A from selected group O sera, when eluted from A1 RBC, will not agglutinate Ax even though the original serum showed an appreciable titre against Ax. The presence of the eluted antigen also suggests an explanation for the behaviour of antibody recovered from the red cells of various subgroups of A towards Ax.

A new reagent (ZZAP) having multiple applications in immunohematology.

A reagent (ZZAP) containing a mixture of 0.1 M dithiothreitol (DTT) plus 0.1% cysteine-activated papain was found to dissociate IgG immunoglobulin from red blood cells (RBC) of patients having a positive direct antiglobulin test (DAT) although this could not be achieved with either chemical alone. In all 67 patients tested, ZZAP treatment of IgG sensitized RBC reduced the strength of the DAT, and in all 52 instances tested, this allowed for accurate Rh phenotyping using slide/rapid tube typing reagents. This included five examples in which spontaneous agglutination that occurred in saline or 6% albumin was eliminated by ZZAP. Thus, all red blood cell typing for Rh-Hr antigens was accomplished using slide/rapid tube reagents making unnecessary the use of saline reactive or chemically modified antisera. Kidd antigen typing is also possible after ZZAP treatment of IgG sensitized RBC. In regard to warm autoabsorption tests, ZZAP treatment of 14 RC samples having a positive DAT proved preferable to heat elution technics since equal or greater amounts of IgG were removed by ZZAP and little or no hemolysis resulted. ZZAP has no effect on ABH, Rh or Kidd antigens but denatures Duffy, MNSs and all Kell antigens tested (K:1-K:7, K:11-K:14, K:18, K:19). This should prove valuable in certain investigations of multiple alloantibodies and, moreover, may allow for better characterization of Kell antigens.

Detection of ABO group-specific substances on the red cells of the cynomolgus monkey.

Several attempts were made to demonstrate the presence of ABO blood group substances on the red blood cells (RBC) of the cynomolgus monkey. Neither the direct hemagglutination test nor the antibody absorption test was successful in detecting these substances from whole RBC, the stromata or the butanol extracts of RBC. By the heat elution of antibodies, however, the presence of ABH substances was demonstrated on RBC of every phenotype without fail. In all cases, there was a reciprocal relationship between the ABH substances of RBC and of saliva and the anti-A and anti-B antibodies in serum, being in good agreement with Landsteiner's rule. Accordingly, it can be concluded that a small amount of ABH substances must be present on RBC of the cynomolgus monkey.

A new method of antibody elution from red blood cells.

A method of antibody elution from red blood cells using xylene is described. It can be completed in 15 minutes after cell washing and requires no special equipment. For warm antibodies, the eluates prepared were more potent than those prepared by Rubin's ether method. It was as effective as Landsteiner and Miller's heat elution method in investigations of ABO hemolytic disease of the newborn. The clinical application of this method in the investigation of hemolytic disease of the newborn, delayed transfusion reaction and autoimmune hemolytic anemia was described.

Elution of HL-A-specific antibodies from platelets.

Abstract. The preconditions and kinetics of absorption and elution of HL‐A specific antibodies from platelets, their antibody binding capacity (‘activity’) and the stability of HL‐A receptors on platelets were studied. It was found that the acid elution technique is the only useful method as compared to the ether or heat elution. The optimal ratio of absorbed serum to eluate volume, platelet number, incubation temperature and time were evaluated. At pH 3.0–3.5 (range tested between pH 1.5 and 5.0) the strongest eluate activity was obtained. The fixation of HL‐A specific antibodies to platelet receptors was not reduced by repeated washings with buffered saline, hypo‐ and hyperosmolaric salt solutions (range 0.077 to 2.464 mol NaCl). The capacity of antigenic receptors of platelets to bind complement‐fixing HL‐A antibodies remained unaltered after treatment with distilled water, hypo‐ and hyperosmolaric salt solutions (range as above) and incubation between pH 5 and 9. Freezing (−20°C), heating (56°C) and short incubation at pH 10 moderately altered HL‐A receptors on platelets, which clearly deteriorated at pH 3 or after incubation at 100°C. A standardized elution procedure is recommended.

Elution of HL-A-Specific Antibodies from Platelets

The preconditions and kinetics of absorption and elution of HL-A specific antibodies from platelets, their antibody binding capacity (‘activity’) and the stability of HL-A receptors on platelets were studied. It was found that the acid elution technique is the only useful method as compared to the ether or heat elution. The optimal ratio of absorbed serum to eluate volume, platelet number, incubation temperature and time were evaluated. At pH 3.0-3.5 (range tested between pH 1.5 and 5.0) the strongest eluate activity was obtained. The fixation of HL-A specific antibodies to platelet receptors was not reduced by repeated washings with buffered saline, hypo- and hyperosmolaric salt solutions (range 0.077 to 2.464 mol NaCl). The capacity of antigenic receptors of platelets to bind complement-fixing HL-A antibodies remained unaltered after treatment with distilled water, hypo- and hyperosmolaric salt solutions (range as above) and incubation between pH 5 and 9. Freezing (-20 °C), heating (56 °C) and short incubation at pH 10 moderately altered HL-A receptors on platelets, which clearly deteriorated at pH 3 or after incubation at 100 °C. A standardized elution procedure is recommended.

Haemadsorption by lymphoid tissue from normal mice.

Tissue sections of normal mouse lymphoid organs were shown to adsorb erythrocytes of sheep and chicken origin. Only the marginal zone of spleen follicles and the subcapsular and medullary sinuses of lymph nodes were re‐active. Sheep and chicken erythrocytes adhered to the same areas and formed mixed clusters when tested together. Haemadsorption by tissue sections was inhibited by anti‐lymphocyte serum and anti‐mouse IgM. It was also abolished by acid or heat elution. Haemadsorption was probably due to cytophilic antibodies of the IgM class adsorbed to tissue macrophages.

Adoptive transfer of neonatally induced tolerance into normal mice

Neonatally induced tolerance in mice was adoptively transferred with living splenocytes into normal immunocompetent syngeneic adult recipients. It was also transferred passively with antisera from tolerant mice into syngeneic neonatal recipients. Immune splenocytes transferred anamnestic responsiveness rather than tolerance. No free antibody was detected by heat elution from tolerant spleen cells, but it was found in a form complexed with antigen and dissociable with acid. These results together with those reported in the accompanying paper on neonatal tolerance suggest a role for antibody in the induction and maintenance of this model of tolerance.

Properties of Specifically Purified Kidney Localizing Antikidney Antibody

Summary Significant amount of material containing 18% of the total nitrogen and 60% of the total phosphorus was solubilized by heating lyophilized sediment of rat kidney homogenate at 60°C at pH 8. The heat extract thus obtained partially neutralized the localizing activity of anti-kidney antibody. Therefore, specifically purified antibody prepared by heat elution of adsorbed antibody on untreated kidney sediment seems to be already neutralized to some extent. Electrophoresis on starch of anti-kidney antibody indicated the γ-globulin nature of localizing antibody. A large portion of the γ-globulin from specifically purified antibody was converted to faster migrating components, probably antigen-antibody complexes, after neutralization with the trypsin digest of kidney sediment. Further evidence for the presence of antigen-antibody complexes in specifically purified antibody was also indicated from the electrophoretic results. Ultracentrifugal studies showed that the localizing antibody is primarily in the light γ-globulin and excluded the possibility that it is significantly present in heavy globulins. The radioiodination and the specific purification by adsorption-elution procedure does not seem to cause any appreciable change in the sedimentation properties of antibody globulin.

ABO isoimmunization: the use of the specific Coombs and heat elution tests in the detection of hemolytic disease

Summary A method of early recognition and identification of ABO erythroblastosis is presented. This involves the use of a potent antihuman globulin serum which has been pretested for its ability to detect adsorbed antibodies of the gamma and nongamma globulin varieties. When a positive Coombs test is found on a sample of blood from the umbilical cord, the infant's cells are treated by the heat elution method for the removal of adsorbed antibodies. The eluates thus obtained are tested against a panel of cells containing known antigens, and the specific antibodies are identified. With this early information, the physician is forewarned of the possible development of clinical disease. In our series, which includes over 316 cases of ABO isoimmunization detected by this approach, it was found that the incidence of ABO isoimmunization approximates that of Rh-Hr incompatibilities. The occurrence of clinical disease in the ABO group was much less frequent, however. Of the 3,921 heterospecific pregnancies to the ABO system, 316 infants showed positive direct Coombs tests. When heat elution tests were done on blood taken from the infant's umbilical cord, 183 or 57.91 per cent were specific for the A antigen, 28 or 8.86 per cent agglutinated only B erythrocytes, and 105 or 33.22 per cent reacted with both A and B red blood cells and appeared to be the anti-C of Wiener.

Organ Specificity of I131 Labeled Rabbit Kidney Eluates in Rats and Rabbits

Summary Rabbits were immunized against rat kidney. On the assumption that the kidneys of these rabbits would have bound antibodies against kidney tissue, the rabbit kidneys were removed, homogenized, washed, and extracted using mild heat (60°C) or acidity (pH 3.2). The eluted material was labeled with I131, further purified, and injected into rabbits and rats to test for preferential localization. Similar preparations were made using normal rabbit kidneys. Preparations from both sources showed preferential kidney localization in both rats and rabbits persisting for days. Localization was greater for preparations made from immunized kidney, but the difference has not been demonstrated to be statistically significant. One preparation of immunized kidney showed a concentration of I131 in the kidney of injected rabbits fifteen times higher than the next highest organ of thirteen organs studied, the spleen, in rabbits sacrificed three days and a week after injection. Three days following injection of another type of labeled preparation into rabbits, 10% of the injected I131 was found localized in the kidneys. Heat eluted and purified labeled eluates of kidneys from the immunized rabbits, but not normal rabbits, localized in rat small intestine as well as kidney following intravenous injection. It proved possible largely to separate the rat intestine localizing component from the kidney localizing component of these preparations. Preparations made in a similar manner using the heat elution techniques from rabbit spleen, striated muscle, adrenal, and lung showed little kidney localizing tendency when injected into rabbits and rats.