Radioimmunoassay Studies of the Cross-reacting Antibody of Human Group O Sera

Cross-reacting antibody was obtained from three recently stimulated group O donors. The biological reactions of cross-reacting antibody were investigated by a study of the binding characteristics of 125I-labelled purified IgG fractions with red cells and by the reactions of whole antiserum or eluates with 125I-labelled blood group glycoproteins. The bulk of cross-reacting antibody activity is restricted to the IgG fraction. Experiments with 125I-labelled IgG and with the radioimmunoassay system revealed that cross-reacting antibody has a higher binding constant than specific anti-A or anti-B. In the radioimmunoassay system complete inhibition of cross-reaction antibody was obtained either with D-galactose or with N-acetyl-D-galactosamine, but the same concentrations of monosaccharides gave only partial inhibition of specific anti-A and anti-B antibodies. The results indicate that cross-reacting antibody binds to a smaller determinant than specific antibody. Investigations with other monosaccharides did not, however, reveal a common inhibitory structure. The ability of cross-reacting antibody to bind to both group A and B antigens may, therefore, be a property of a 'polyfunctional' antibody binding site.     

Defining the specificity of anti-A/B IgM/G antibodies with different antigens and lectins

The specificity of anti-histo blood group A/B antibodies is defined by the antigen used for their production and selection (in the case of mouse monoclonal antibodies, mAbs) or by the antigens present on various intestinal bacteria (for polyclonal human IgM and/or IgG, phAbs). Absorption experiments with red blood cells and free antigen have been used earlier to define specificity; here we use A/B substances of human, animal and synthetic origin as well as known reactivities of mAbs or lectins as tools to further characterize epitope properties of the A/B antigens. The signal response patterns obtained with identical phAbs anti-A/B IgM/G on three different antigens are superposable. In contrast, commercially available mAb anti-A/B IgM, when tested on the same antigens, revealed different response curves. The chemical specificity of lectins for distinct monosaccharides in terminal position was exploited to delineate the specificity of mouse ascites anti-A IgG1 and of phAbs anti-A/B IgM/G. Helix pomatia lectin inhibited the access of MB9 to porcine A almost completely whereas binding capacities of human anti-A IgM/G were inhibited by 50 and 62%, respectively. In similar experiments Bandeiraea simplicifolia lectin was seen to inhibit access of anti-B IgM/G to horse B by 34 and 58%, respectively. No inhibition was seen with lectin from Ulex europaeus or concanavalin A on plates coated with the three different A or B substances. Thin-layer chromatography (TLC) of antigens revealed one spot for the synthetic trisaccharides, whereas the human and animal blood group substances showed 2 and 3 spots, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Reactions of IgG and IgM Anti-A and Anti-B Blood Group Antibodies with 125I-Labelled Blood Group Glycoproteins

Abstract. The reactions between IgG and IgM anti-A and anti-B and ¹²⁵I-labelled A and B blood group glycoproteins were investigated by a modification of the Farr technique. Inhibition curves obtained in a radioimmunoassay system indicate that IgM anti-A and anti-B antibodies have a significantly higher binding constant for A and B glycoproteins than IgG anti-A and anti-B. The observed difference in binding constant may explain the fact that A and B glycoproteins readily inhibit agglutination of A and B red cells by IgM anti-A and anti-B but less readily inhibit agglutination by IgG anti-A and anti-B.     

Further Evidence for the Presence of A Antigen on Group B Erythrocytes through the Use of Specific Exoglycosidases

Certain antibodies have shown the ability to detect small amounts of A antigenic structures on certain group B cells. These rare cells that reverse group as B, are designated here as B(A) cells. Among the anti-A antibodies capable of detecting these cells are MHO4 (an IgM murine monoclonal antibody) and polyclonal anti-A derived from blood group O donors. The latter (anti-A, B) have been adsorbed exhaustively with normal B cells, to deplete the serum of antibodies to group B antigen. The cell specificity detected by these antibodies can be removed only by an alpha-N-acetylgalactosaminidase (A-zyme) but not by an alpha-galactosidase (B-zyme). Inhibition studies show that these reactions can be inhibited by A secretor saliva and cannot be inhibited by B secretor saliva. Moreover, papain treatment of normal group B cells not previously agglutinable with these antibodies, now causes these cells to become reactive, and this specificity, too, is removed only by A-zyme. These results suggest that low levels of blood group A antigen are being recognized by these antibodies and that these structures can exist not only on B(A) cells but on all group B erythrocytes.     

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.     

A Mouse Monoclonal Antibody with Anti-A,(B) Specificity Which Agglutinates Ax Cells

A hybridoma (ES-15) was obtained by fusing the NS-1 cell line with spleen cells from a mouse immunised with soluble blood group A2 substance. The cloned hybridoma culture supernatant was shown to contain an IgM class antibody which strongly agglutinates group A cells and weakly agglutinates group B cells. The serological specificity of this antibody is described as anti-A,(B) in this report. The abilities of unconcentrated monoclonal anti-A,(B), a commercial human polyclonal anti-A,B (group O serum) and a commercial monoclonal anti-A reagent to detect 15 examples of Ax cells were compared by both slide and tube techniques. Using a slide technique monoclonal anti-A,(B) agglutinated 14 examples of Ax cells, human anti-A,B 2 examples, while monoclonal anti-A failed to detect any of the Ax cells tested. Similar differences in the reactivity of the three antibodies were observed using a tube technique. Data are also presented which show that a 1:1 (v/v) mixture of monoclonal anti-A,(B) with a monoclonal anti-B reagent is an effective replacement for human anti-A,B in ABO grouping procedures.     

Specific and cross-reacting antibodies in ABO heterospecific twin pregnancy

A "study in depth" is reported concerning the case of hemolytic disease of agroup B infant born to a group O mother, whose group A twin was apparentlyunaffected. It was shown that the hemolytic disease of the affected infant wasdue to a specific anti-B antibody. The study included parallel examinations ofthe antibodies in the sera of the three individuals.

The specificity of the B-anti-B reaction was demonstrated in vitro and in vivo.The powerful anti-B antibody of the mother had no effect on the group A twin,in whose serum anti-B was present in large amounts.

In vitro, studied by the usual technics of cross absorption the maternaland the fetal anti-A and anti-B serum antibodies behaved as if strictly specific.By applying successive absorption, elution and neutralization techniques, however, it was possible to demonstrate additional cross-reacting antibodies in thematernal serum which could be separated from one another and from the specificanti-A and anti-B antibodies. From the erythrocytes of the normal group Atwin such cross-reacting antibody could be eluted.

The cross-reacting anti-B antibody, isolated in pure form in eluates, could beshown to be loosely attached to group A erythrocytes without producing visibleagglutination reactions while after elution from A cells it did visibly agglutinategroup B cells. It could be eluted from A cells, absorbed by fresh A cells and reeluted while retaining its anti-B effect. It was neutralized by group B saliva only.A separate anti-A antibody with similar properties was eluted from B cells andspecifically neutralized by group A saliva.

A partial affinity of these antibodies for heterologous erythrocytes but not forspecific soluble substances was thus demonstrated. These findings support neitherthe linkage hypothesis of cross reactions between anti-A and anti-B nor theC-anti-C hypothesis of hemolytic disease. They are in keeping with the view thatgroup O sera contain variable complexes of anti-A and anti-B antibodies, composed of multiple fractions with different partial specificities. It is suggested thatthe occurrence or non-occurrence of cross-reacting antibodies found in sera ofgroup O mothers whose infants develop hemolytic disease is best explained onthis basis. It is further stressed that the demonstration of an antibody in motheror child in ABO hemolytic disease does not necessarily indicate its pathologicsignificance.

Submitted on January 11, 1957 Accepted on April 19, 1957     

Further Observations on the Serological Specificity C of the A-B-O Blood Group System

S ummary : *A simple method has been described for producing antisera containing anti-C antibodies of high titre and avidity, by immunizing group O individuals having low titres of isoagglutinins with porcine blood group substance A. Such injections can stimulate a rise not only of the anti-A titre but also of anti-C with no noticeable effect on the anti-B titre. The only disadvantage is that although the suitably diluted reagents are potent, they contain anti-A in addition to anti-C, and therefore can be used for testing for the presence of C only those red cells or other materials which lack group A specificity. However, reagents suitable for testing for C in the presence of A could be prepared by immunizing group O subjects with group B red cells or B group substance.
Evidence is presented that the specificity C is shared by all red cells and other materials having A-like and or B-like specificities, though rare individuals exist having red cells agglutinable by anti-C though lacking both A and B. It is suggested that the specificity C is not due to a determinant group shared by A and B group-specific substances, but, instead, that overall similarities in the surface structure of their molecules are responsible. Analogies are pointed out which are comparable to the relationship of factor C to the A-B-O system; namely, of U to the M-N-S and rh^G to the Rh-Hr system, and also of C^c to the simian C-E-F blood system of chimpanzees.
* To avoid ambiguity, symbols for blood factors (serological specificities) of red cells and their corresponding antibodies are printed in boldface type, symbols for genes and genotypes are printed in italics , and symbols for agglutinogens, phenotypes and blood group systems are printed in regular type.     

Spectrin autoantibodies in normal human serum and in polyclonal blood grouping sera

Naturally occurring spectrin autoantibody was detected in normal human sera and in polyclonal blood grouping sera by the immunoblotting technique. The autoantibody seems to be IgG, stable, of high titre and low affinity. It was detected in all sera tested. Preparations of monoclonal antibodies directed against some blood group antigens and anti-A1 lectin reagent were devoid of spectrin autoantibody as expected. This autoantibody may be instrumental in clearing red cell membrane components from the circulation during haemolysis. Care must be exercised in studies designed to determine the association of some blood group antigens with the red cell membrane skeleton, when using polyclonal sera which contain spectrin autoantibodies in addition to the antibodies against the blood group antigen in question.     

The Pathogenesis of ABO Haemolytic Disease of the Newborn

Summary. A study of the pathogenesis of ABO HD is essentially a study of the interrelationships of the maternal IgG anti-A/anti-B antibodies with the various components of the 'protective mechanism'.
There is an equilibrium of IgG antibodies across the placenta and thus, ABO HD occurs more frequently in cases with high titre IgG antibodies than in cases with low titre IgG antibodies.
The foetal blood group substance 'protects' the infants by competing with the red cells for the incompatible antibodies. Small amounts of the homologous blood group substance reduce the IgG anti-A/anti-B reaction with A/B red cells and the degree of inhibition increases as the antigenic strength of the red cells decreases.
Possible reasons for the difference in combining power of the various adult and foetal red cells are discussed.
A small deficiency of non-secretor infants and fathers was observed and the possible roles of secretor status in ABO HD are discussed.     

A Detailed Serological Study on the Prediction and Diagnosis of ABO Haemolytic Disease of the Newborn (ABO HD)

Summary. An evaluation is given of haemolysin, neutralisation, thermostability, saline titration, anti-A^p antibody absorption and 2-mercapto-ethanol studies on the sera from 178 clinically affected ABO HD cases and 130 control cases.
The antiglobulin titre on the 2-mercapto-ethanol treated sera which determines the IgG titre was the best procedure used for the demonstration of significant immune anti-A/B antibodies in the maternal sera.
The screen series on 5,704 sera from group 0 mothers for the detection of immune anti-A/B and the attempted prediction of ABO HD revealed that ABO HD occurs with a frequency of about 0.8%. Exchange transfusion was needed in six cases i.e. about 0.1% of all the infants of group O mothers screened.
The minimum criteria for the diagnosis of ABO HD are the serological demonstration of incompatible anti-A/B antibodies in an eluate prepared from the baby' red cells, accompanied by the clinical observation of jaundice, or more rarely pallor due to anaemia, in the infant during the first few days following birth.
In the absence of elution or serum studies on a blood sample from the baby, the presence of an incompatible high titre (>256) IgG antibody in the maternal serum provides good presumptive evidence that the baby may be suffering from ABO HD.     

Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens.

It is widely accepted that the serum of healthy individuals contains natural antibodies only against those blood group A or B antigens that are not expressed on the individual's red blood cells. The mechanisms involved in tolerance to autologous blood group antigens remain unclear. In the present study, we show that IgM and IgG antibodies reactive with autologous blood group antigens are present in the immunoglobulin fraction of normal human serum. Natural IgG anti-A antibodies purified by affinity chromatography from IgG of individuals of blood group A exhibited an affinity for A trisaccharide antigen in the micromolar range and agglutinated A red cells at sixfold higher concentrations than those required for agglutination with affinity-purified anti-A IgG of individuals of blood group B. Whereas autoantibodies reactive with self A and B antigens are readily detected in purified IgG and IgM fractions, their expression is restricted in whole serum as a result of complementary interactions between variable regions of antibodies. These observations suggest that tolerance to autologous ABO blood group antigens is dependent on peripheral control of antibody autoreactivity.     

Characterization of rabbit polyclonal sera against recombinant Shiga toxin and its subunits for detection of Stx-producing E. coli

Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera could also neutralize the cytotoxicity of the shiga toxin on vero cells. The neutralizing ability of the prepared sera was compared for different subunits. The neutralization of toxicity was observed by incubation of raised sera with the rStx or Shiga toxin from wild type strains. The inhibition of cell toxicity was shown by anti-A, anit-B and anti-AB antisera, separately. It was shown that anti-A antibody, more significantly recognized Stx producing strains, comparing to anti-B antibody. These sera from immunized rabbits were also used as specific antibodies in Enzyme-Linked Immunosorbant Assay (ELISA) for detection of Shiga toxin. It was demonstrated that the raised antibodies especially antibody against A subunit could be a useful tool for immunological diagnosis of STEC induced infection.     

Serological Studies on some Freshwater Fishes: Examination for ABH Erythrocyte-Specific Serum Agglutinins

Abstract. Haemagglutination studies showed that sera of some species of freshwater fishes contained high titres of agglutinins with specificities directed toward the ABH blood group antigens. The significance and potential usefulness of serological studies with these freshwater fishes is discussed with particular reference to: (1) the possibility of using certain fish sera as anti-A, anti-B, cross-reacting anti-A and B and anti-H blood group reagents, and (2) the potential of some freshwater fish species as laboratory animals for the production of anti-carbohydrate antibodies.     

Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library

BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens. We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens. MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen. Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells. Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA). RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated. CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens.     

Sensitive methods for determining subclasses of IgG anti-A and anti-B in sera of blood-group-O women with a blood-group-A or -B child

The determination of the subclasses of IgG antibodies against blood groups A and B is important in order to improve our understanding and predict haemolytic disease of the newborn due to IgG anti-A or -B. We describe two techniques that circumvent the problem of the agglutination of A and B red cells by the corresponding IgG antibodies in saline: an antiglobulin consumption test and a modified solid-phase micro-immunofluorescence test. The results of the two techniques are compared with the results obtained in the indirect antiglobulin test beyond the saline agglutination titre in a microplate technique. The solid-phase micro-immunofluorescence test was the most sensitive for the determination of the subclasses of IgG anti-A and -B. Usually sera contained IgG2 anti-A, B in a higher titre than antibodies of other subclasses.     

The red cell antigens A, B, D, U, Ge, Jk3 and Yta are not detected on human granulocytes

We report the inability to detect the following red blood cell antigens on human granulocytes: A, B, D, U, Gerbich (Ge), JkaJkb (Jk3) and Cartwright (Yta). To study each antigen, granulocytes were purified on density gradients, fixed in glutaraldehyde, and the uptake of specific antisera measured using two direct immunological techniques: 125I-staphylococcal protein A (125I-SPA) binding and avidin-biotin-complex (ABC) immunoperoxidase staining. Glutaraldehyde fixation was shown not to affect the antigenicity when the antisera were tested using red blood cells. Using three anti-A, three anti-B and three anti-A,B antisera, our 125I-SPA results of 47 tests with granulocytes from group A individuals and 39 tests with granulocytes from group B individuals indicate that A or B antigens are not expressed on human granulocytes. Tests using ABC were also negative with 37 and 36 granulocytes from group A or B individuals, respectively. In addition, no positive results using 125I-SPA were obtained with granulocytes from individuals having antigen positive red cells when tested with two anti-D (number of tests performed (n = 22), three anti-Ge (n = 22), three anti-U (n = 20), two anti-Jk3 (n = 17), and three anti-Yta (n = 25); control anti-NA1 or -NB1 antisera were invariably positive. Also, using these antisera, no positive results were obtained by ABC except with one anti-Yta antiserum which was positive with one of seven granulocytes tested. This anti-Yta was also positive with three of 10 granulocytes by 125I-SPA. This activity was shown to be due to a granulocyte-specific antibody; adsorption of the antiserum with human granulocytes removed all activity against granulocytes but did not reduce the activity against red cells. Thus, our results are in agreement with recent reports which demonstrated the absence of the A, B and D antigens on human granulocytes. However, we have been unable to confirm previous reports which indicated the presence of the U, Ge and Jk3 antigens on human granulocytes. Also, we have been unable to detect the Yta antigen on human granulocytes.     

Isoprecipitins: Precipitating Antibodies Reacting with Blood Group Specific Substances

A precipitating iso-anti-A was discovered in the serum of a female blood donor when routine tests were being performed for anti-Au HAA antibody by means of counter-immunoelectrophoresis. The isoprecipitin reacted with the sera of blood group A and AB donors and with dilute solutions of blood group A substance. Using the same technique, a significantly higher instance of anti-A and anti-B isoprecipitins was found in mothers of children suffering from haemolytic disease of the newborn due to ABO incompatibility. Regular screening of antenatal sera for precipitating anti-A and anti-B is recommended for possible antenatal prediction of affected infants. False positive reactions might be obtained when known Au HAA antigen-positive sera used for screening for the presence of human anti-Au HAA also contain A or B specific substances which could react with corresponding precipitating antibodies.     

Maternal antibodies against fetal blood group antigens A or B: lytic activity of IgG subclasses in monocyte-driven cytotoxicity and correlation with ABO haemolytic disease of the newborn

IgG antibodies against blood group antigens A or B (anti-A/B) are able to sensitize erythrocytes for destruction in an antibody-dependent cell-mediated (ADCC) assay with monocytes as effector cells. The activity of maternal IgG anti-A/B in this test was compared with clinical signs of haemolytic disease of the newborn (HDN). When the ADCC was negative (less than 10% of the sensitized cells lysed), signs of increased red-cell destruction in the children were never observed. In three cases with a strongly positive ADCC (greater than 45% lysis), the children were severely affected and needed more than one exchange transfusion. In the cases with greater than 10% but less than 45% lysis in the ADCC, there was no clear correlation between the result of the ADCC and the degree of lysis in the newborn infants. In these cases, the degree of lysis of the red cells of the infant was shown to be strongly influenced by the number of A/B antigens per red cell. There was a direct correlation between the degree of lysis in the ADCC and the titre of IgG3 anti-A/B in the sera. There was comparable activity of maternal IgG anti-A/B in the ADCC test in the 32nd week of pregnancy and at the moment of delivery.     

Epitope Specificity of Blood-Group-A-Reactive Murine Monoclonal Antibodies

Monoclonal antibodies (MABs) towards blood groups A manifested three broad patterns of binding distinguished on the basis of affinities for A1 or A2B phenotype red blood cells and blood group substances. Competitive inhibition of binding of radiolabelled MABs by other unlabelled antibodies and absorption studies with synthetic haptens suggested that GpA specificity was expressed as two topographically related structures, one being the terminal GpA trisaccharide and the second probably involving part of the oligosaccharide backbone. The enhancement of binding of an extremely low-affinity antibody by higher affinity antibodies recognising either epitope indicated a topographic, possibly conformational relationship between these epitopes. It is suggested that only those antibodies that recognise terminal GpA trisaccharides can agglutinate weak A2B cells.