Rapid Purification of Anti-I and Anti-i Cold Antibodies by Affinity Chromatography

A method is described for the rapid purification of serologically active high titer anti-I and anti-i cold antibodies from the sera of patients with chronic cold agglutinin disease (CCAD). The purification procedure is based on thermal affinity chromatography, using desialated orosomucoid (alpha 1-acid glycoprotein)-Sepharose 4B conjugated beads. The nature of the interaction between the cold agglutinins (CA) and the desialated orosomucoid is unknown. Inhibition studies, however, revealed that the cold hemagglutinating activities of all the anti-i sera were inhibited by desialated orosomucoid while only 1 out of 4 of the anti-I sera was similarly affected. Anti-I or anti-i antibodies were separated from whole sera in 7 out of 7 samples with a recovery in most cases of 100% of the cold hemagglutinating activity. The resultant products were purified monoclonal IgM fractions which could react with anti-kappa and anti-mu but not with anti-lambda sera. The homogeneity, purity and specificity of all preparations were confirmed by immunodiffusion analysis against purified I and i blood group antigens isolated from human erythrocyte membranes, zonal and right-angle electrophoresis and hemagglutination or hemagglutination inhibition studies.     

Reappraisal of the Role of Anti-i in Haemolytic Anaemia in Infectious Mononucleosis

S ummary . Current literature implies that haemolytic anaemia in infectious mononucleosis is regularly caused by the temporary production of high thermal amplitude cold agglutinins of anti-i specificity. More recently, Capra et al (1969) suggested interaction between IgG anti-i and anti-IgG antibodies as the cause of haemolytic anaemia in infectious mononucleosis. A detailed serologic evaluation of three patients during moderate to severe haemolytic anaemia in infectious mononucleosis revealed high thermal amplitude anti-i in only one. This patient's direct antiglobulin test (DAT) was negative using anti-IgG but was 3 + using anti-C₃ and -C₄. The serum antibody titre against cord or adult Oi cells was 512 at 4°C and 4 at 31°C. The anti-i was inhibited by mercaptoethanol, and anti-IgG was not found in the patient's serum. Patient 2 had a negative DAT and patient 3 had a 2 + DAT using anti-C₃ and anti-C₄. Anti-i was present only in low titre at 4°C and was unreactive at 20°C. It was inhibited by mercaptoethanol. These patients' sera contained anti-IgG antibodies. In none of our patients were warm autoantibodies detected. This data demonstrates that haemolytic anaemia in infectious mononucleosis is not necessarily associated with high thermal amplitude anti-i. Further, since the anti-i antibodies in patients 2 and 3 were of low titre, were inhibited by mercaptoethanol, and did not react at physiologic temperatures, the mechanism of haemolysis in these patients does not seem related to the interaction of anti-IgG and IgG anti-i antibodies. Further work is necessary to clarify the mechanism of haemolytic anaemia in infectious mononucleosis.     

The role of macromolecular components in anti-gamma, alpha, mu, kappa, lambda, C3 and C4 antiglobulin sera

Summary . Studies are described of the early and late responses of rabbits immunized with human IgG protein. Early macroglobulins (19S) antiglobulin antibody is compared with 7S antibody in tests against red cells strongly and weakly coated by IgG anti-Rh(D) antibody. The 19S antibody, when reacted on a glass plate, is slower to produce maximum agglutination than 7S antibody, is enhanced by 4% human serum albumin, and is of at least equal sensitivity in detecting very weakly sensitized erythrocytes. The 19S antibody shows no loss of potency during 1 yr storage at - 20°C, but is unstable beyond 6 mth storage at 4°C. The 19S antibody is non-precipitating against its specific antigen, which complicates assessment of its specificity. It is shown that admixture of 19S with 7S antiglobulins alters the manifestation of the prozone characteristic of the 7S antibody alone. Macroglobulin antiglobulin reagents raised against human γ, α, μ, K, λ, C₃ and C₄ antigens are compared with 7S reagents of similar specificities in tests against coated cells from 14 patients with acquired autoimmune haemolytic anaemia (AAHA), three patients receiving methyldopa, and against normal cells sensitized with anti-D, anti-Fy^a, anti-Kell, anti-Le^a and anti-Jk^a isoantibodies. The results put macroglobulin antiglobulin reagents in perspective in relation to the broad field of antiglobulin testing.     

Autoimmune hemolytic anemia in association with monoclonal IgM(kappa) with anti-i activity.

A patient with a warm autoimmune hemolytic anemia with an immunoglobulin G (IgG) panagglutinin, also had monocional IgM(kappa) cold agglutinin with anti-i activity. Ninety per cent of the peripheral blood lymphocytes had surface immunoglobulin and the number of T cells was diminished. A subpopulation of the patient's lymphocytes formed rosettes with cord (i) erythrocytes and not with adult (l) erythrocytes. The finding of increased lymphocytes bearing i-binding sites and a monoclonal antibody with anti-i activity could be related to shared idiotypic determinants between antigen-binding sites and serum antibody. The occurrence of two autoantibodies in this patient suggests an immune regulatory disorder.     

Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens.

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B-cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline-encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti-I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.     

Non Hodgkin’s lymphoma presenting as neutropenia related to an IgM monoclonal anti-i antibody

A sixty-year old female was referred to the Internal Medicine Department for the treatment of a diffuse high-grade non Hodgkin’s lymphoma. She presented episodes of fever in a context of neutropenia (neutrophils 0.35 × 10⁹/l from 1.6 × 10⁹/l white blood cells). Hemoglobin level was 8.2 g/dl and platelets 132 × 10¹²/l. A monoclonal IgM-(kappa) protein (48 g/l) was detected in her serum. A direct antiglobulin test on the red cells proved positive with anti-C3d but not with anti-IgG antiglobulin, due to the presence of an IgM cold antibody with a serological anti-i specificity. The IgM antibody was found on the patient’s neutrophils as well as in her serum. This antibody recognized all neutrophils tested in conventional serological tests whatever the neutrophil phenotypes in systems NA, NB, and 5. It was demonstrated that it recognized the i antigen expressed on the neutrophils. These results suggest that a cold agglutinin anti-i might be responsible for neutropenia in some patients.     

Cold Haemagglutinins Associated with Splenomegaly in New Guinea

Summary. A high incidence of cold haemagglutinins with anti-i specificity was found in serum samples from patients with massive splenomegaly of uncertain cause in the Watut valley of New Guinea. The samples contained increased concentrations of macroglobulin, 16–33% of which represented cold agglutinin activity. Tropical splenomegaly constitutes another condition in which anti-i appears in association with hyperplasia of reticulo-endothelial tissue.     

Monomeric (7S) immunoglobulin M antibodies to hepatitis delta virus in hepatitis type D

The pattern of the immunoglobulin M antibody to the hepatitis delta virus distinguishes acute from chronic hepatitis D. Expression of the immunoglobulin M antibody to the hepatitis delta virus is relatively weak and short-lived in self-limited hepatitis but strong and persistent in chronic forms. To study the nature of the immunoglobulin M antibody to the hepatitis delta virus in acute hepatitis D and in chronic hepatitis D, antibody-positive sera were submitted to rate zonal centrifugation to separate monomeric 7S from pentameric 19S immunoglobulin M antibodies. Sera were from 6 patients with acute self-limited hepatitis, 4 patients with chronic hepatitis D, and 6 patients with hepatitis D progressing to chronicity. The immunoglobulin M reactivity was measured by a specific immunoassay based on capture of mu-chains by anti-mu linked on a solid phase. Only 19S antibody was found in acute hepatitis D. In contrast, all patients with chronic hepatitis D circulated 7S antibody in addition to the 19S antibody. In patients with progressive hepatitis D, both the 7S and 19S antibody variants were present at the onset of the disease. The difference in the antibody response between acute hepatitis D and chronic hepatitis D is not only temporal and quantitative but also qualitative. The expression of 7S antibody seems to be an immunologic event specific for chronic hepatitis D.     

Identification of normal B-cell counterparts of neoplastic cells which secrete cold agglutinins of anti-I and anti-i specificity

A monoclonal antibody, which recognizes a cross-reacting idiotypic determinant present on human cold-reactive autoantibodies with anti-I or anti-i binding activity, has been found to specifically inhibit the cold agglutination of red cells. This suggests that an epitope close to the binding site of such autoantibodies is being recognized. The antibody has been used to identify tumour cells in the blood of three patients with cold haemagglutinin disease, and to analyse the heterogeneous nature of the neoplastic B-cell clone present in the bone marrow of one of the patients. Using S-phase analysis, it was found that cell proliferation was occurring in the bone marrow but not in the blood, and that the major proliferating population was that of lymphoplasmacytoid cells containing large vesicular inclusions of idiotypic IgM. It has also been possible to locate normal B-cells which are recognized by the anti-idiotypic antibody. Such cells have been found throughout the normal adult lymphoid tissue where they account for 2.9-10.8% of the B lymphocyte population. They are also present in fetal spleen at 15 weeks gestation, indicating that immunoglobulins bearing this sequence form part of the immature B-cell repertoire.     

Hereditary Erythroblastic Multinuclearity Associated with a Positive Acidified-Serum Test: a Type of Congenital Dyserythropoietic Anaemia

In five patients (including two sisters) an unusual anaemia was characterized by erythroblastic multinuclearity, ineffective erythropoiesis and a positive acidified-serum test. Unlike PNH, the sugar-water test was always negative in these patients. The lysis of their cells by acidified normal sera indicated their abnormal sensitivity to an agglutinating and complement-binding antibody present in some normal subjects. The patients cells gave high agglutination scores with anti-i, and were unusually susceptible to lysis by anti-i and anti-I. The disorder is apparently inherited as an autosomal recessive character.     

Li Cold Agglutinin: A Further Antibody Recognizing Sialic Acid-Dependent Antigens Fully Expressed on Newborn Erythrocytes

The cold agglutinin anti-Li recognizes a sialic acid-dependent antigen fully expressed only on newborn red cells. Although it resembles anti-i in all aspects except for its nonreactivity with neuraminidase-treated red cells, it is entirely different from anti-i, since sialic acid is not involved in i antigenic determinants.     

Diagnosis of Haematological Disease using Anti-i

Leukaemic blast cells were obtained from the blood of six patients with acute lymphoblastic leukaemia (ALL) and 15 patients with acute myeloblastic leukaemia (AML). The blasts were compared with lymphocytes from normal subjects in cytotoxicity and 125I-labelled antibody binding tests using several examples of anti-i. As much i antigen was detected on ALL blasts as on normal lymphocytes; much less i antigen was detected on AML blasts. Studies of three patients with morphologically undifferentiated acute leukaemia suggest that, in tests with anti-i, blasts from such patients react either like lymphoblasts or myeloblasts despite the absence of the corresponding morphological features.     

Light chain heterogeneity of 19S and 7S anti-gamma-globulins in rheumatoid arthritis and subacute bacterial endocarditis.

Both 19S and 7S anti-gamma-globulins in rheumatoid arthritis sera are enriched in kappa light chain bearing antibody molecules when compared to total 19S and 7S globulins from the same individuals. In patients with subacute bacterial endocarditis 19S anti-gamma-globulins are also, to a degree, enriched in kappa light chains, whereas the 7S anti-gamma-globulins have kappa to lambda light chain ratios indistinguishable from total 7S globulin.     

Light chain heterogeneity of 19s and 7s anti-γ-globulins in rheumatoid arthritis and subacute bacterial endocarditis

Both 19S and 7S anti-γ-globulins in rheumatoid arthritis sera are enriched in kappa light chain bearing antibody molecules when compared to total 19S and 7S globulins from the same individuals. In patients with subacute bacterial endocarditis 19S anti-γ-globulins are also, to a degree, enriched in kappa light chains, whereas the 7S anti-γ-globulins have kappa to lambda light chain ratios indistinguishable from total 7S globulin.     

Suppression of chemotactic activity of human polymorphonuclears by monoclonal IgMs with and without biological activity

The influence of monoclonal IgMs on migration of human polymorphonuclears was studied at various temperatures by the use of 19 sera with monoclonal IgMs from patients with macroglobulinemia of Waldenstrom (MW) without obvious biological activity, 29 sera with monoclonal IgM cold agglutinins (18 with anti-I and 11 with anti-i IgMs) and 3 sera with monoclonal IgM rheumatoid factor (RF). Under-agarose migration method and modified Boyden chamber method with double filters and 51Cr-PMNs were used. In under-agarose method, chemotactic differentials for controls, MW, anti-I, and anti-i groups were, respectively, 57 +/- 8 mm, 39 +/- 9 mm, 44 +/- 14 mm, and 32 +/- 16 mm at 37 degrees C and 47 +/- 18 mm, 22 +/- 11 mm, 17 +/- 9 mm, and 15 +/- 12 mm at 24 degrees C. All three sera with IgM RF inhibited chemotaxis. The differences between all groups and controls were significant at p less than 0.01. Random migration was inhibited at 24 degrees C (p less than 0.01) but not at 37 degrees C. Inhibitory concentrations of IgM in the sera tested were equal or less than 0.5 mg/ml. Thirteen sera were tested by the modified Boyden chamber method. At 37 degrees C 8 of 13 sera and at 24 degrees C 11 of 13 sera inhibited significantly chemotaxis at a concentration of IgM of 1 mg/ml. The lowest inhibitory concentration of IgM was 25 micrograms/ml. Eleven chromatographically pure IgMs were tested in the under-agarose assay. At concentrations of 0.4-3.7 mg/ml, eight IgMs inhibited chemotactic differential at 37 degrees C and nine inhibited it at 24 degrees C. At concentrations of 0.6-2.0 mg/ml, all seven pure IgMs tested by the Boyden chamber method significantly inhibited chemotaxis at 24 degrees C and 37 degrees C. Some IgMs inhibited chemotaxis at concentrations as low as 25 micrograms/ml. Ten IgM CA were eluted from the red blood cells. Eluates inhibited strongly chemotaxis at 24 degrees C and 37 degrees C. Heat inactivation did not alter inhibitory activity of IgM, however pepsin digestion or reduction and alkylation of purified IgMs did abolish their inhibitory activity. Inhibition of chemotaxis was not related to the light chain type, the titre, or the thermoamplitude of cold agglutination. However, monoclonal IgMs with anti-i cold agglutinin activity were stronger inhibitors than anti-I. Since 75% of IgMs tested inhibited chemotaxis at 37 degrees C, it is possible that monoclonal IgMs, especially those with anti-i cold agglutinin activity, inhibit PMN migration in vivo.     

Fatal cold anti-i autoimmune haemolytic anaemia complicating hairy cell leukaemia

Hairy cell leukaemia (HCL) is a rare lymphoproliferative disorder associated with pancytopenia, splenomegaly and the presence of typical hairy B lymphocytes in the bone marrow and/or peripheral blood. The most significant complication relates to opportunistic infections that arise as a consequence of neutropenia and monocytopenia. HCL is occasionally associated with systemic autoimmune disorders including polyarteritis nodosa and rheumatoid disease. Secondary autoimmune haemolytic anaemia (AIHA) appears to be rare. We report on two cases of HCL complicated by fatal cold anti-i AIHA. Fulminant haemolysis causing death is rare in cold AIHA and only a few individual cases have been reported, none having anti-i specificity.     

Clinical significance of two forms of IgM antibody to hepatitis delta virus

Separation of 7-8 S and 19 S forms of serum IgM antibodies to the hepatitis delta virus by rate-zonal centrifugation was carried out on serum from 24 patients with hepatitis delta virus infection: 4 patients with acute, self-limited hepatitis; 5 patients with hepatitis delta virus superinfection progressing to chronicity; and 15 patients with chronic hepatitis delta virus. The high molecular weight IgM form (19 S) was predominantly detected in acute hepatitis delta virus cases, whereas the low molecular weight (7 S) form was found in chronic hepatitis delta virus cases. The serological profile of these two forms of IgM antibody to hepatitis delta virus was investigated in serial samples from five patients with acute hepatitis delta virus superinfection that evolved to chronic hepatitis delta virus. We found that, in the acute stage of the disease, the 19 S form was predominant, whereas 6 mo later a predominance of 7-8 S IgM was observed. These results suggest that IgM antibody to hepatitis delta virus antibody forms are different in acute and chronic hepatitis delta virus infection and that their detection only helps in differentiating an acute infection from a chronic infection but not a hepatitis delta virus-hepatitis B virus-HBV coinfection from hepatitis delta virus superinfection in the acute stage of the disease.     

Auto-immune haemolytic anemia complicating infectious mononucleosis in a patient with hereditary elliptocytosis.

Auto-immune haemolytic anemia complicating infectious mononucleosis occurred in a patient with hereditary elliptocytosis. A cold antibody of IgM anti-i specificity with narrow thermal amplitude was identified in the serum and the erythrocytes were found to be coated with complement. Significantly excessive erythrophagocytosis was demonstrated in samples of the patient's blood which had been chilled and then incubated at 37 degrees C. The patient recovered spontaneously. The elliptocytosis does not appear to have contributed to the episode of haemolytic anaemia; the other elliptocytic member of the family (her father) had no history and no present evidence of haemolysis.     

Striking inverse correlation between IgG anti-F(ab')2 and autoantibody production in patients with cold agglutination

Previous experiments showed that the physiologic IgG anti-F(ab')2 antibody suppresses the response of human autoreactive B cells. In the present study, we analyzed the IgG anti-F(ab')2 antibody in 293 patients with cold agglutination (CA). Their average IgG anti-F(ab')2 titer was not much different (211 +/- 8.3) from that of 279 healthy persons (195 +/- 6.7). However, CA patients with high anti-F(ab')2 titers had low CA autoantibody titers and vice versa (P = .0028; rho = - 0.175). The stratification of patients according to the auto-antibody's specificity (anti-I, anti-i, anti-Pr) showed an inverse correlation between anti-F(ab')2 and CA in the anti-I group (P = .0057; rho = - 0.180). Interestingly, the association was present only in patients whose disease was caused by noninfectious agents (P < .0001; rho = - 0.423). The inverse correlation argues for an important role of the IgG anti-F(ab')2 in the regulation of autoantibody production in CA patients.