The Separation and Antigenic Characteristics of Some Fractions of Papain Digests of Human Serum Gamma Globulins, and the Antigenic Relationships of Some Human Gamma Globulins with the Gamma Globulins of Some Other Mammalian Sera

1. Papain digests of human γ globulin (7S γ globulin) containing slow (S) and fast (F) immunoelectrophoretic components were fractionated by chromatography on diethylaminoethyl cellulose and by salting out with ammonium sulphate. No direct correlation was found between the antigenic characteristics and the chromatographic and electrophoretic properties of the S component fractionated by these methods.

2. β₂M globulin (γ₁ macroglobulin or 19S γ globulin) contained the S component of γ globulin, but the F component was not detected.

3. Rhesus monkey γ globulin more readily absorbed the anti-F antibodies than the anti-S from anti-human γ-globulin sera. Both rhesus and digested human γ globulins showed a stage of inhibition, when they completely prevented the precipitation of intact human γ globulin by its antiserum. Rhesus γ globulin, however, showed a reaction of partial identity (spur formation) on Ouchterlony analyses with human γ globulin and its antiserum, whereas digested human γ globulin showed a reaction of complete identity. The significance of these observations is discussed.

     

Human Reagins to Grass Pollens and Moulds: Their Purification and Physico-chemical Characterization

Chromatography of reaginic sera (to grass pollens and to moulds) on DEAE-cellulose leads to the concentration of reagins in three well separated fractions. They emerge (a) together with siderophilin, (b) with the early albumin-containing fractions, and (c) in association with strongly adsorbed proteins that require a comparatively high ionic strength buffer for their elution. The proportion of reagins in each of these fractions varies with different sera and with small alterations in the experimental procedure. All three reaginic fractions contain small amounts of γ globulins (referred to as R globulins) of mobilities slightly less than that of siderophilin; (c) contains much α_2M globulin and there are traces of α_2M in (a) and (b). Yet the bulk of the γ globulins are shown to be free from reaginic activity, and the same is true of the α_2M and the β_2M globulins, of siderophilin and of albumin. The purer the reaginic fractions are the smaller is the portion of the reagins that can be precipitated with the γ globulins by half saturation with ammonium sulphate. In contrast to the bulk of the γ globulins, R globulins and reagins appear to associate readily with other serum proteins, particularly with α_2M globulins. Fractionation with sodium sulphate produces three fractions of similar potency although they have little in common; one consists of the bulk of the γ globulins (0–15 per cent w/v), the most active fraction of the remaining globulins (15–18 per cent) and the third fraction (supernatant from the 18 per cent precipitate) of albumin containing some α globulins, but only a trace of γ globulin.

Ultracentrifugation studies on three main reaginic DEAE-fractions show that the bulk of the reagins are not macroglobulins although misinterpretation can arise from complex formation with α_2M globulin.

High agglutination titres (Stavitsky's method, Stavitsky and Arquilla, 1958) for pollen proteins are associated only with the unretarded non-reaginic γ globulins of post-treatment sera (which contain the blocking antibodies) although traces of agglutinating antibodies can be demonstrated in many fractions, including the reaginic fractions derived from the sera of untreated hay fever subjects.

     

Modulation of Fc receptors on human peripheral blood lymphocytes by antisera against β2 microglobulin, Ia or immunoglobulin

The association between Fc receptors and other surface molecules was examined by EA-rosette (EAR) inhibition experiments. Twenty to 30% EAR were detected in suspensions of peripheral blood lymphocytes from normal individuals. Anti-β₂ microglobulin (βMi) sera fully suppressed EAR whereas anti-Ig, anti-Ia sera and heat aggregated IgG inhibited only 50–60% EAR. Thus almost half of the detected EAR were apparently not surface Ig positive B cells. Rabbit and monkey anti-βMi sera suppressed EAR effectively whereas rat and chicken antisera, despite strong βMi binding capacity, inhibited EAR poorly. The latter result was ascribed on the basis of immunofluorescent analysis to inadequate capping of βMi. Incubation of PBL with whole antisera suppressed EAR to a similar degree at either 0° or 37°, whereas F(ab′)₂ fragments were inhibitory only at 37°. Taken together the results suggest that Fc receptors can be inhibited by antibodies with specificity against any cell surface antigen. The blocking mechanism may be due to steric hindrance by the Fc part of antibody molecules and/or F(ab′)₂ fragment mediated co-capping.     

Changes in serum alpha2 globulins in reticuloses

The total concentrations of α₂ globulin and two of its components, haptoglobin and caeruloplasmin, have been determined in the sera from 78 patients with primary malignant diseases of lympho-reticular tissue (reticuloses). The total α₂ globulin fraction was significantly raised only in Hodgkin's disease. The serum haptoglobin was also significantly increased in Hodgkin's disease and some other reticuloses, including acute leukaemia, lymphosarcoma, and reticulum cell sarcoma. The serum level of caeruloplasmin was raised in all reticuloses. There was possibly some correlation between the rise in haptoglobin and caeruloplasmin concentrations and the total α₂ globulin in Hodgkin's disease but this was not statistically significant. The significance of these results is discussed, and it is concluded that the increase in α₂ globulin which occurs in Hodgkin's disease is due to an increase in several of its components and not to a single component.     

Laboratory evaluation of anti-phospholipid syndrome: a preliminary prospective study of phosphatidylserine/prothrombin antibodies in an at-risk patient cohort

Immunoglobulin (Ig)G/IgM autoantibodies to phosphatidylserine/prothrombin (aPS/PT) were evaluated individually and in combination with criteria anti-phospholipid (aPL) tests in a prospectively ascertained cohort of patients at risk for anti-phospholipid syndrome (APS). One hundred and sixty (160) consecutive requests for lupus anti-coagulant (LAC) from the University of Utah Health Sciences Center were identified during 8 weeks. Of these, 104 unique patients had additional requests for cardiolipin (aCL) and/or beta2 glycoprotein I (aβ₂GPI) IgG and/or IgM; samples were retained and analysed for aPS/PT, aCL and/or aβ₂GPI IgG and IgM antibodies. Following testing, a comprehensive chart review was performed and patients categorized according to their clinical diagnosis. Individual and combined sensitivities, specificities, odd ratios (OR), diagnostic accuracy for specific tests or combinations by receiver operating characteristic (ROC), area under the curve (AUC) analyses and correlations between test results were determined. The sensitivities of aPS/PT IgG/IgM (54·6/45·5%) were lower than LAC (81·8%) but higher relative to aCL IgG/IgM (27·3/0%) or aβ₂GPI IgG/IgM (27·3/0%). The best correlation between LAC and any aPL test was observed with aPS/PT (P = 0·002). There was no significant difference in the diagnostic accuracies for any panel with LAC: LAC/aβ₂GPI IgG/aCL IgG [AUC 0·979, OR 475·4, 95% confidence interval (CI) 23·1–9056·5, P = 0·0001 and LAC/aβ₂GPI IgG/aPS/PT IgG or LAC/aPS/PT IgG/aCL IgG (AUC 0·962, OR 265·3, 14·2–4958·2, P = 0·0001). The high correlation between LAC and aPS/PT IgG/IgM in this preliminary study suggest that this marker may be useful in the evaluation of APS. More studies to determine the optimal aPL antibody tests combination are needed.     

An Investigation of Methods of Isolation of β2M1 Globulin (Syn.: Iota Protein, 19S γ Globulin, γ1 Macroglobulin, β2M Globulin) and its Association with Isoagglutinin Activity, together with Preliminary Observations on Other Macroglobulins of Slow Electrophoretic Mobility in Normal Human Serum

An investigation is described of methods of isolation and purification of the high molecular weight protein (here called β_2M1 globulin) which is often associated with the isoagglutinin activity of normal human serum. In the course of these experiments it was observed that ultracentrifugal concentration of this protein from serum fractions gives rise to the simultaneous concentration of two other macroglobulins (here called β_2M2 and β_2M3 globulins).

It was found that the concentration of β_2M1 globulin in normal serum is about 25–50 mg. per 100 ml. In serum from the blood of normal donors of groups A, B and O, isohaemagglutinin activity is associated with the β_2M1 globulin but probably accounts for 1 per cent or less of the total β_2M1-globulin concentration in serum. This activity may represent the so-called `natural' isohaemagglutinin.

The immunological relation is discussed of the normal β_2M1 globulin to certain other members of the `family' of immune globulins (γ globulin and β_2A globulin) and to the pathological macroglobulins occurring in Waldenström's syndrome.

Although rabbit antibody to β_2M1 globulin cross-reacts with γ globulin, suggesting that a portion of each molecule bears antigenic groupings in common, it was found that a considerable degree of β_2M1-globulin specificity could be demonstrated after absorbing antisera with purified γ globulin. Attention is also drawn to the evidence that deficiency of either β_2M1 globulin or γ globulin can occur independently, suggesting different cellular sources of origin.

The pathological macroglobulin demonstrable in Waldenström's syndrome is closely related immunologically to normal β_2M1 globulin but is often deficient in isoagglutinin activity. This immunological relation suggests the use of specific anti-β_2M1-globulin antiserum as a simple means of distinguishing macroglobulinaemia from myelomatosis or other conditions with raised levels of γ globulin.

The β_2M2 and β_2M3 globulins are only demonstrable in ultracentrifugal concentrates of normal serum. Evidence is presented to suggest that these proteins are nevertheless present as trace components of normal serum. The solubility and electrophoretic characteristics of these proteins resemble those of β_2M1 globulin, but they can be distinguished by immunological methods from β_2M1 globulin and each other. Attention is drawn to the evidence that β_2M2 and β_2M3 globulins may also be increased in the serum of some cases of macroglobulinaemia.

     

A comparison of tests for thyroglobulin antibody

A comparison is made of tests for thyroglobulin antibody, using gel diffusion, electroprecipitin, bentonite flocculation, tanned red cell agglutination, and latex slide agglutination techniques on sera from cases of Hashimoto's disease and other thyroid disorders. Any increase in γ globulin was also noted from the serum electrophoretic pattern. The gel diffusion and electroprecipitin tests are shown to be comparable in their sensitivity, as are the bentonite flocculation and tanned red cell agglutination tests. The flocculation and agglutination tests were oversensitive. The latex slide test in conjunction with the electro-precipitin test is recommended for routine use in the detection of Hashimoto's disease.     

Kinetic studies on phagocytosis IV. Cellular defects and humoral inhibition as causes of impaired neutrophil phagocytosis in sarcoidosis

Kinetic measurements of the serum-independent uptake of IgG-coated or complement-opsonized latex particles have been performed in 58 patients with sarcoidosis. The mean rate for phagocytic uptake of IgG particles was 0·56 min^-1 which was not different from that of the controls (0·59 min^-1). The phagocytosis of complement-opsonized particles was in the patient group 0·53 min^-1 and significantly (P<0·001) reduced compared to the rate of the controls (mean rate 0·94 min^-1), indicating neutrophil C3b-receptor dysfunction in sarcoidosis. PMNs from patients with sarcoidosis were not stimulated by the presence of autologous serum in contrast to PMNs from normals and in individual cases even a reduced uptake was found. More than one-third of the sarcoid sera also inhibited the phagocytosis of normal PMNs indicating the presence of a phagocytosis-inhibitory activity in sarcoid sera. Patients with more severe lung affection as estimated by measurements of total lung capacity, central airway obstruction, small airway function and pulmonary X-ray changes had a more reduced PMN phagocytosis in the presence of autologous serum than those with minor signs of lung affection (P<0·05). The phagocytosis-inhibitory activity of sarcoid serum was also more pronounced in those individuals who had high pulmonary score (P<0·05) or radiographic stage II-IV sarcoidosis (P<0·01). No correlation was found between serum levels of lactoferrin or lysozyme and any of the phagocytic variables while elevated β₂-microglobulin levels were associated with more pronounced serum-mediated inhibition of PMN phagocytosis (P<0·05). The relevance of these findings to the pathogenesis of granuloma formation in sarcoidosis is discussed.     

Antigenic Analysis of Rheumatoid Factor

Papain digestion of human γ globulin yields two main antigenic components, of slow (S) and fast (F) electrophoretic mobility. Recent work has shown that β₂M globulin (γ₁ macroglobulin) contains S but not F antigenic groupings. It also contains specific groupings which may be called X. Rabbit antisera have been prepared which are specific for S, F and X.

Sheep red cells, `sensitized' with rabbit anti-sheep cell serum as for the Rose-Waaler test, were incubated with rheumatoid arthritis sera. Under appropriate conditions these cells failed to agglutinate when resuspended in saline, but could be agglutinated by anti-γ globulin (S+F), anti-S and anti-X, but not by anti-F. Cells incubated with normal serum failed to agglutinate. Thus rheumatoid arthritis sera contain a protein possessing the antigenic characteristics of β₂M globulin which specifically coats sensitized sheep cells. This provides further evidence of the antibody nature of rheumatoid factor.

     

The effects of ALG on the murine immune response to sheep erythrocytes

Antilymphocyte globulin (ALG), and to a lesser extent normal rabbit globulin (NRG), when given to mice prior to immunization with sheep-RBC suppress both the γM and γG_2a responses. Globulin injected after the antigen suppresses the γG_2a response, augments the γG₁ response and has little effect on the γM response. These effects are also observed in mice partially paralysed to rabbit γ globulin. In another system—the response to hapten—protein conjugates precursors of antibody producing cells were found to be more resistant to ALS treatment in vivo than were helper cells. It is concluded that the suppressive effects of ALG treatment are largely due to the direct action of ALG on helper cells (T-cells). The mechanism of the adjuvant-like effect is unclear.     

The Significance of Multiple Antibody Components in Serum of Immunized Rabbits

The electrophoretic patterns of six sera from rabbits immunized by two or more courses of intravenous injections of killed pneumococci type III showed multiple peaks in the γ-globulin region. Such sera contained large amounts of antibody (up to 85 per cent of the total γ globulin) against the capsular polysaccharide. One serum contained a cryoglobulin, which contained almost as great a proportion of specific antibody as did the remaining γ globulin.

The electrophoretic patterns and antibody contents were similar in the water-soluble and water-insoluble fractions of γ globulin.

The sedimentation constant and diffusion coefficient of a water-soluble fraction of γ globulin, containing 85 per cent specific antibody, were measured. The values, at 0.4 per cent protein concentration, were S_20.w = 6.97×10^-13 and D_20.w = 4.16×10^-7 cm.² sec.^-1, corresponding to molecular weight 159,000.

The antibody-containing globulin from one serum was separated by zone electrophoresis into three fractions with different electrophoretic mobilities. These contained 53–71 per cent of antibody precipitable by type III pneumococcus capsular polysaccharide. Only doubtfully significant differences were found in respect of amino-acid composition, hexose and hexosamine contents, or antigenic characteristics.

A method was devised for detecting small amounts of antibody against capsular polysaccharide by means of red cells sensitized with culture filtrates of capsulated pneumococci.

The antibody was also fractionated by chromatography on anion-exchange cellulose, and numerous fractions with antibody activity were obtained. It was shown by labelling the γ globulin with ¹³¹I that similar fractionation occurred both in the presence and absence of other serum components. All the chromatographic fractions of γ globulin were found to contain approximately similar proportions of antibody. By electrophoresis in starch gel the fractions were found to differ from one another and to be heterogeneous.

The implications are discussed of the finding that antibody against type III pneumococcus capsular polysaccharide can occur over the entire range of γ-globulin molecules.

     

Human anti-immunoglobulin antibodies with specificity for native and pepsin-digested IgD

When human sera were tested against red cells coated with IgD by the CrCl₃ technique, agglutinating activity was found in a high proportion of sera from patients with systemic lupus erythematosus and rheumatoid arthritis, while sera from normals and patients with non-rheumatoid diseases contained only trace activity. Haemagglutination inhibition experiments indicated that the activity was directed against the Fc part of IgD, and reduction with 2-mercaptoethanol, sucrose density gradient ultracentrifugation, and absorption experiments all indicated that the agglutinating activity was due to IgM antibodies. In some sera a very weak activity was also found against antigens revealed by pepsin digestion of IgD.After density gradient ultracentrifugation of sera at pH 3·0, the 19S fractions showed higher antibody activity against IgD than fractions obtained at pH 7·2, indicating that the sera contained complexes of IgD and anti-IgD. Agglutination inhibition experiments with different IgD myeloma proteins or whole myeloma sera did not give evidence for subclasses or genetic polymorphism of IgD.     

Further attempts to characterize the normal incomplete cold antibody

Attempts at eluting the normal incomplete cold (n.i.c.) antibody from sensitized red cells or red cell stroma, using standard methods, were unsuccessful; thus the antibody could not be detected in the eluates obtained by heating at 56° or 37° or by dissociation at acid pH. The n.i.c. antibody was partially eluted from sensitized red cells only when elution was carried out at 37° into serum instead of saline.

Elution of the antibody from dextran (Sephadex G-200)—n.i.c. antibody complex formed at 0° was also achieved using a 15 per cent NaCl solution at 37°.

Since it has been shown that red cells sensitized with the n.i.c. antibody are not agglutinated by anti-γG, anti-γA or anti-γM-globulin sera, an attempt at producing an antibody specifically reacting with the n.i.c. antibody was made by injecting into a rabbit the eluate obtained from zymosan—n.i.c. antibody complex. The immune serum was found to inhibit the n.i.c. antibody activity when added to normal serum and to interfere with the property of normal serum requiring the properdin system.

In the present work it is also confirmed that the antibody is not associated with γG or γM-globulin; thus adult and cord sera and one serum from a patient with severe hypogammaglobulinaemia were fractionated with zone electrophoresis or DEAE-cellulose chromatography and it was found that the antibody was not present in the fractions containing the bulk of γG or γM-globulin.

     

Antibodies to β2 microglobulin in the sera of patients with systemic lupus erythematosus

The present study reports the detection of antibodies to β₂ microglobulin in the sera of patients with systemic lupus erythematosus (SLE). Using a Farr-type ammonium sulphate precipitation assay, test sera were reacted with ¹²⁵Iβ₂ microglobulin, and immunoglobulins precipitated by 50% saturated ammonium sulphate. Increased β₂ microglobulin binding activity (normal values: mean±2 sd = 35.5 ±7.8) was detected in 18 of 42 SLE sera. Anti-HLA sera did not reveal increased binding activity, suggesting that the antibody in SLE serum was directed toward free β₂ microglobulin. Direct validation was done by reacting ¹²⁵Iβ₂ microglobulin with 4 SLE sera having increased ¹²⁵Iβ₂ microglobulin binding activity, and subjecting the reactants to sucrose density gradient ultracentrifugation. Two peaks were obtained, one corresponding to free β₂ microglobulin, and the other to 7S material complexed to β₂ microglobulin. Normal sera demonstrated only one peak corresponding to unbound β₂ microglobulin. Assays of β₂ microglobulin binding activity on protein fractions obtained by Sephadex G200 column chromatography also showed the presence of increased binding activity with 7S fractions. Using a double antibody assay, the 7S material reactive to β₂ microglobulin was demonstrated to be IgG. It was also shown that sera with abnormal β₂ microglobulin binding activity had higher titres of antinuclear antibody compared to those lacking such activity (t = 3.18; P<0.01), indicating the pathogenetic relationship of this antibody to increased disease activity. This antibody may be responsible for some of the abnormalities of cell-mediated function previously described in SLE patients.     

Effect of Antibody on Heterologous and Homologous Cells in Tissue Culture

In order to compare antibody against heterologous and homologous tissue in the same serum, rabbits were immunized with rat kidney and complete adjuvant. The resulting sera showed antibody against both rat and rabbit kidney. Cultures of rat kidney cells were killed by exposure to these sera. A concentration of 0.3 per cent γ globulin (ammonium sulphate fraction) was adequate to kill cultures in the absence of complement, but smaller concentrations were effective when guinea-pig complement was added. The cell surfaces were shown to have taken up antibody by the fluorescent antibody technique. Cytoplasmic staining could only be shown in cells which had previously been injured by freezing and thawing or by fixation. Rabbit kidney cells in culture were unaffected when exposed to whole rabbit anti-rat serum, but were killed and their cell membranes stained on exposure to γ globulin derived by (NH₄)₂SO₄-fractionation from such serum and having the same complement-fixation titre as the parent serum. At least 0.6 per cent γ globulin had to be added to kill rabbit cell cultures. It was found that normal rabbit serum had a partial protective effect against this antibody. Fractionation of sera by gradient centrifugation or chromatography on DEAE-cellulose showed that while antibody against heterologous tissue was found both in the 7S γ globulin and macroglobulin fractions, antibody against homologous tissue was confined to the latter. It is considered that the findings do not support a concept of an in vivo pathogenic role for circulating antibody.     

The Occurrence of Haemagglutinating Antibody to Penicillin

Certain sera from individuals receiving large doses of penicillin have been found to contain a factor which can cause agglutination of human red cells sensitized with penicillin. Such sera were detected by a preliminary white tile screening technique and subsequently titrated in tubes.

The haemagglutinating factor appeared to be heat stable (56.0° for 6 hours at least) and active over a pH range of approximately 4.8 to 9.0. In the presence of complement certain strongly positive sera caused haemolysis of the red cells. The addition of penicillin inhibited the haemagglutination reaction but this effect was reversible by the addition of penicillinase. Certain agglutinating sera gave positive complement fixation tests but no visible precipitates were obtained by tube or double diffusion agar gel techniques. Attempts to obtain active eluates from agglutinated sensitized cells were unsuccessful. It was also found that the haemagglutinating factor did not appear to have any inhibiting effect on the bactericidal action of penicillin.

Starch-gel electrophoresis of positive sera demonstrated haemagglutinating activity in two pre-albumin zones. One such zone, pre-albumin-2, has been shown to be acidic-alpha-1-glycoprotein whereas the nature of pre-albumin-1 remains obscure. In twenty-two of twenty-three sera so tested agglutinating activity was confined to these two zones. In the remaining serum, in addition to activity in the two pre-albumin zones, positive agglutination of sensitized cells was also brought about by the gamma-globulin fraction, and this serum was the only one which gave a positive antiglobulin test. The interpretation of these electrophoretic findings is not altogether clear and requires further elucidation.

     

IgA/IgC cryoglobulinaemia with vasculitis

A mixed IgA/IgG cryoglobulin complex was found in the serum of a 61-year-old man suffering from rheumatoid arthritis, Raynaud's phenomenon and vascular purpura. The purified complex was progressively insoluble at temperatures below 37°C. Reversible loss of cryoprecipitability was seen in conditions of extreme pH, in concentrated urea solutions and in 2-mercaptoethanol. Inactivation of complement at 56°C did not affect cryoprecipitability. The complex contained no complement and showed no anticomplementary activity. Analytical ultracentrifugation of the cryoglobulin at 37°C showed 7S and 11S components present in equal concentration.

The component immunoglobulins of the complex were separated by anion exchange chromatography at 37°C. Crossmixing studies with purified normal immunoglobulins indicated that the patient's IgA component was essential for cryoprecipitability of the complex. RA latex tests for anti-IgG activity at 37°C showed strong agglutination with both cryoglobulin complex and the isolated IgA component; this protein was found to be monoclonal with type K light chains.

Vasculitis, induced by skin testing the patient with his own plasma and isolated cryoglobulin was found to be histologically indistinguishable from that occuring spontaneously; reduction of symptoms paralleled reduction of cryoglobulin on treatment. These observations strongly support the hypothesis that the cryoglobulin complex is responsible for the patient's vasculitis.

     

Identification of the Components of Complement Participating in the Antiglobulin Reaction

Red cells sensitized with a complement-binding antibody and then incubated with fresh serum have been shown to be coated with β_1C and β_1E globulin, two components of the complement system that have been isolated recently. Red cells presumably in the state EAC′_1,4 reacted with anti-β_1E, and cells presumably in the state EAC′_1,4,2,3a reacted with anti β_1E and with anti-β_1C. Agglutination of complement-coated cells by `broad spectrum' antiglobulin sera was effectively inhibited by purified β_1E and β_1C globulin. Red cells from certain patients with acquired haemolytic anaemia were found to be coated in vivo with β_1E and β_1C globulin. The function and significance of the two serum components have been discussed.     

Classification of Blood-Group Antibodies as β2M or γ Globulin

Thirty selected blood-group antibodies (excluding anti-A and anti-B) have been classified as β₂M (19S γ) globulin, γ (7S γ) globulin or mixtures, using the following three methods: fractionation on a DEAE-cellulose column; indirect anti-globulin tests, using specific anti-β₂M-globulin and anti-γ-globulin sera; and treatment with 2-mercapto-ethanol. With only minor exceptions, results obtained with the three methods were in agreement.Most blood-group antibodies within the Le, MNSs and P systems appear to be `naturally occurring'' and these were found to be β₂M globulin. Blood-group antibodies within the Rh, K and Jk systems, which had arisen after an antigenic stimulus, were usually γ globulin but were occasionally β₂M globulin.Antibodies composed of β₂M globulin usually behave as agglutinins but may behave as incomplete antibodies (e.g. some examples of anti-Jk^a); conversely, antibodies composed of γ globulin usually behave as incomplete antibodies but may behave as agglutinins (e.g. an example of anti-M).The ability to bind complement seems to be related more to the blood-group specificity of the particular antibody than to its molecular size. For example, anti-Jk^a, when composed either of γ or β₂M globulin, seems invariably to bind complement, whereas potent anti-M or anti-Rh, whether composed of γ or β₂M globulin, do not bind complement.     

Anti-globulin antibodies detecting hidden γG antigens: characterization of the `pepsin site' by different immune antisera

The antigenic region of human γG-globulin revealed by pepsin digestion has been studied by means of four different `pepsin agglutinator' (PA) containing sera. Two of these were `naturally occurring' agglutinators found in human and subhuman (baboon) sera respectively. The other two were heteroimmune antisera produced by the immunization of rabbits with human and baboon F(ab′)₂ fragments. All of the PAs were agglutinating but not precipitating.

Each PA reacted specifically with γG₁ and γG₃ immunoglobulins as determined by testing with isolated human myeloma proteins. One of the rabbit PAs (RAHPA) reacted with both reduced and unreduced pepsin fragments in contrast to the other agglutinators. In addition, different reaction patterns were evident when the PAs were tested with a panel of pepsin digested primate γG-globulins.

The general trend of the data suggested that a similar antigen was being detected by the four PAs. Further, it was evident that a limited degree of heterogeneity existed within the human `pepsin site' and between similar regions in higher primates.