Studies on globulin and antibody production in mice thymectomized at birth

Serum globulin levels and antibody against sheep red cells, Salmonella typhi (`O' and `H'), haemocyanin and pneumococcus type 3 capsular polysacharide (SSS III) were studied in C3H/Bi and C57BL×C3H/Bi F₁ hybrid mice thymectomized within 18 hours of birth. Thymectomized mice tend to recover more slowly than intact mice from the physiological hypo-γ-globulinaemia present 3–4 weeks after birth, but the majority of mice that survived beyond 6 weeks had serum globulin levels comparable to those in intact animals. Increased γ_1A-globulin levels were observed in some thymectomized mice which survived for 7 weeks or more and the immuno-electrophoretic patterns sometimes resembled those seen in mice with γ_1A myelomas. Macroglobulin (γ_1M) was detected in the sera of thymectomized mice which contained normal amounts of γ-globulin.

Total serum protein levels of groups of thymectomized and intact mice were not significantly different. The turnover rate of plasma albumin was also closely similar in both groups, but that of γ-globulin was accelerated in most of the thymectomized mice examined.

Antibodies against SSS III, sheep red cell agglutinins and typhoid `H' agglutinins behaved as 7S globulins, in contrast with sheep red cell haemolysins which belonged to the 19S group.

Antibody levels following primary stimulation with sheep red cells and Salmonella antigens were usually, but not always, much lower in thymectomized mice than in intact control mice. However antibody levels against haemocyanin and SSS III were within the range of controls in more than half the thymectomized mice. Few thymectomized mice after stimulation with a mixture of three antigens failed to give a detectable antibody response to all the antigens, and some responded as well as did intact mice.

The significance of these findings is discussed in relation to current theories of the function of the thymus in the development of immune responses.

Many of the older thymectomized mice showed a characteristic wasting syndrome, but it is unlikely that a general failure of antibody response can account for this. Evidence for the presence of auto-antibodies against red cells or nuclear or cytoplasmic constituents was sought but was not found.

     

Effect of Transplantable Plasma-Cell Tumours of Antibody Response in Mice

The response to primary antigenic stimulation was measured in C₃H mice bearing two types of plasma-cell tumours, the 5563 tumour producing γ-type myeloma proteins and the 5674 tumour producing β-type myeloma proteins. Haemocyanin and pneumococcus type III polysaccharide, which produce 6.5S antibodies, and sheep erythrocytes, which produce 18S haemolysins, were used.

Levels of serum antibody to all antigens were low in mice with the plasma-cell tumours, compared with those in normal mice, or mice with a mammary carcinoma. The larger the tumour, the lower were the antibody levels. Antibody levels were lower generally with the 5563 tumours than with the 5647 plasma-cell tumours of similar weight.

Although rapid catabolism of normal gamma globulins contributes to the low level of serum antibody in mice with the 5563 γ-type tumour, diminished responsiveness of the antigen-susceptible cells appears to be the principal factor in the impaired antibody response in mice with the 5647 β-type plasma-cell tumour.

     

Immunological Identification of the Pathological Cold Auto-Antibodies of Acquired Haemolytic Anaemia as β2M-Globulin

Cold antibodies were separated from the sera of six patients suffering from the cold-haemagglutinin syndrome and from one patient with acquired haemolytic anaemia secondary to lymphosarcoma by dissociation of the specific antigen-antibody complexes. The eluted antibodies were studied (a) by immuno-electrophoresis along with the parent sera against horse anti-human serum and (b) by double diffusion in agar gel along with electrophoretically separated `γ<sub>1</sub> globulin'† against anti-19S `γ-globulin' rabbit sera.

The protein forming the cold antibody was localized in the β₂-M position on immuno-electrophoresis in each instance. It was found by double diffusion in agar gel to be immunologically identical with the protein forming the abnormal `γ₁-globulin' electrophoretic peak in the parent serum.

The results of these experiments indicate that the cold antibodies derived from patients with the cold-antibody type of acquired haemolytic anaemia are macro-molecular globulins.

     

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.

     

Study of the Antigenic Constituents of Sera from Mouse/Rat Chimaeras

The sera of CBA mice lethally irradiated and restored with rat bone marrow or foetal liver have been examined by immunoelectrophoresis at various times after irradiation. Such sera contain several specific rat proteins including γ globulin, at least two α₂ globulins, one or two β₁ globulins and one β₂ globulin. A large number of specific mouse proteins are also present, but no mouse γ globulins have been detected.

The rat constituents appear progressively and persist for long periods; this implies that they are synthesized by rat cells.

There is a close correlation between the presence of rat γ globulin in the serum and the presence of dividing rat cells in the bone marrow and spleen.

The fact that γ globulin is solely of rat specificity does not necessarily rule out the possibility of immune activity by the host against the graft.

Several of the normal serum proteins, in addition to the γ globulins, originate from the injected cells, and are therefore presumably not synthesized by the liver.

In chimaeras grafted with rat skin, the number of specific rat serum proteins is often larger than the number in chimaeras which have not been grafted.

     

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.

     

in vitro : Interference of Non-specific Gamma Globulins with Passive • Anaphylactic Sensitization of Isolated Guinea-Pig Intestine

Non-specific γ globulins, when added to the incubating medium containing rabbit anti-egg albumin antibody, have been found to impede passive in vitro sensitization of the isolated guinea-pig ileum. Although this interference has been observed with the various γ globulins studied, there is a consistent difference in the intensity depending upon their zoological origin. When rabbit antibody is used for sensitization, the impeding effect of the γ globulins decreases in the following order: rabbit>man>dog>guinea pig>rat>horse>cattle>pig>chicken>goat.

A certain degree of interference has been observed even with horse, cattle, chicken, and goat γ globulins, although it is impossible to passively sensitize the guinea pig with the antibodies originating from those animal species in vivo.

The results reported here are difficult to reconcile with the hypothesis that the non-specific γ globulins interfere with the sensitization process by `competing' with the antibody for the same specific receptors. They rather suggest that the impeding effect of the γ globulins is due to their interaction with the antibody by some physico-chemical process, producing thus an alteration of its specific properties.

     

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.

     

The passive transfer of delayed hypersensitivity in the guinea-pig: II. The ability of passively transferred antibody to cause local inflammation and retention of antigen and the role of these phenomena in the passive transfer of delayed hypersensitivity

It is known that serum increases the passive transfer of delayed hypersensitivity to certain antigens, such as bovine γ-globulin, in the guinea-pig. This synergic effect of serum in the passive transfer of delayed hypersensitivity to bovine γ-globulin was partly, but not completely, produced by serum to haemocyanin when a mixture of bovine γ-globulin and haemocyanin was used for skin testing. It was concluded that part of the synergic action of serum was due to a local inflammatory reaction.

The ability of serum to cause local retention of antigen in the skin was studied using antigens labelled with radioactive iodine. Immune serum favoured the local retention of antigen. The passive transfer of antiserum to bovine γ-globulin, egg albumin and haemocyanin specifically increased the retention of antigen two- to twelve-fold. This ability of serum to cause the local retention of antigen at the site of intradermal injection was present in serum taken 3 weeks after immunization with bovine γ-globulin in Freund's complete adjuvant but absent in serum taken at 1 week. Antiserum also altered the distribution of antigen at the skin site. Autoradiography showed that it increased the area over which an appreciable concentration of antigen occurred.

Active immunization with bovine γ-globulin had a slight effect on the total amount of antigen retained in the skin after intradermal injection. It had a greater effect on the distribution of antigen. In control guinea-pigs 87 per cent of the total amount of bovine γ-globulin retained at 20 hours was found within a radius of 6.5 mm of the centre of injection. In contrast in guinea-pigs immunized with bovine γ-globulin in Freund's complete adjuvant 43 per cent was found beyond this radius. A similar change in the distribution of human serum albumin was seen in guinea-pigs immunized with bovine γ-globulin when the albumin was mixed with bovine γ-globulin. This indicated that factors other than the formation of immune precipitates were sometimes responsible for the local retention of antigen. The total amount of purified protein derivative of tuberculin (PPD) retained and its distribution in the skin was uninfluenced by immunization.

It was concluded that the synergic effect of immune serum on the passive transfer of delayed hypersensitivity was due in part to some aspect of the inflammation caused by antibody antigen reaction and in part to the local retention of antigen caused by antibody.

     

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.

     

On the Sensitizing Properties of Some Normal and Pathologic Human Immune Globulins and Fragments Obtained by Papain or Pepsin Digestion

Although normal γ₁A (β₂A) globulins, myeloma proteins, as well as Bence-Jones proteins and normal human urine γ globulins cross-react with 7S γ globulins, none of these proteins can sensitize the skin of the guinea-pig to give a reverse passive cutaneous anaphylaxis reaction. The 3.5S fragments of human antisera to horse serum proteins can no longer give passive cutaneous anaphylaxis reactions, but fragment C and probably A can inhibit the passive cutaneous anaphylaxis reaction of the original antiserum. By reverse passive cutaneous anaphylaxis, negative results were obtained with each of the three fragments or the 5S pepsin digest.     

Passive sensitization of skin and lung by guinea-pig immunoglobulins

Guinea-pig γ₁- and γ₂-globulins have been purified by preparative electrophoresis followed by chromatography. No γ₁-globulin was detectable in purified γ₂-globulin, but purified γ₁-globulin always contained fast γ₂-globulin. Normal guinea-pig serum contained much less γ₁-globulin than immune serum. Antisera prepared against normal guinea-pig serum did not contain useful amounts of antibody specific for γ₁-globulin.

Guinea-pig lung tissue was sensitized by very low concentrations of guinea-pig γ₁-globulin (of the order of 6×10^-10 molar) but γ₂-globulin antibodies were almost inactive. No evidence was found that the trace of activity in γ₂-globulin was not due to very slight contamination with γ₁-globulin antibodies.

The finding that γ₁-globulin antibodies are far more potent than γ₂-globulin antibodies in sensitizing skin has been confirmed, but several lines of evidence suggest that γ₂-globulin antibodies may also have weak activity. Thus quantitative passive cutaneous anaphylaxis (PCA) tests showed that whenever the γ₂-globulin fraction contained antibody it appeared far more potent relative to γ₁-globulin than when the same proteins were tested on lung tissue. The PCA activity of moderate amounts of purified γ₂-globulin antibodies disappeared faster than the skin sensitization produced by small amounts of γ₁-globulin antibodies, and the γ₂-globulin preparations did not contain enough γ₁-globulin impurity to account for their PCA activity. No inhibition of skin responses was observed with the largest doses of antigen tested.

The most plausible explanation of these results is that, under the conditions of our experiments, γ₂-globulin antibody had weak PCA activity. Objections to this hypothesis are discussed. The PCA activity of γ₂-globulin antibody probably involves a mechanism different from that of the sensitization produced by the highly potent γ₁-globulin antibody.

     

Rat 7S immunoglobulins: characterization of γ2- and γ1-anti-hapten antibodies

Two antigenically distinct populations of rat antibodies to the 2,4-dinitrophenyl group (DNP) have been isolated by chromatography on DEAE-cellulose and named fraction 1 and fraction 3. Both fraction 1 and fraction 3 had sedimentation coefficients of 6.9S, similar hexosamine contents (1.04 and 0.89 per cent) and electrophoretic mobilities similar to the γ₂- and γ₁-immunoglobulins of guinea-pigs and mice. Fraction 1 consisted of two distinct antibody populations with very similar mobilities.

Only fraction 1 was able to sensitize homologous rat skin for passive cutaneous anaphylaxis (PCA). Both preparations were active in haemolysis of passively sensitized sheep red cells in the presence of complement, and fraction 3 was more lytic than fraction 1. In other tests such as PCA in mice, passive haemagglutination and precipitation in gel the behaviour of the two fractions was similar.

Fraction 3 is probably the immunoglobulin designated γA in earlier reports. The results indicate that fraction 1 and fraction 3 correspond to the 7S γ₂- and 7S γ₁-immunoglobulins of guinea-pigs and mice, except for their behaviour in homologous PCA and in passive haemolysis, and that fraction 1 and fraction 3 are rat 7S γ₂- and 7S γ₁-immunoglobulins, respectively.

     

γ1-Antibodies in guinea-pigs infected with the cattle lungworm

The parameters of a system for testing both actively acquired and passive immunity to the cattle lungworm in the guinea-pig are described. It was found that the passive transfer of guinea-pig immunoglobulins conferred a strong immunity. The globulins responsible for immunity were present in a serum fraction containing electrophoretically fast moving globulins and anaphylactic activity. Guinea-pigs were not protected by artificial immunization with adult worm homogenate even though precipitating and anaphylactic antibodies were produced. Artificial immunization may have failed because γ₁- and γ₂-antibodies were produced against irrelevant antigens or because blocking γ₂-antibody was produced in excess.     

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.     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Studies on the Mechanism of Hypersensitivity Phenomena: V. Antigen-Antibody Interaction in the Guinea-Pig Small Intestine*†

A series of exploratory studies have been described in which an isometric apparatus has been applied to the study of the smooth muscle response to immune reactions.

Data are given for the dose-response relationships with several immune systems.

Evidence is provided which relates the length of the induction period to the quantity of antigen and magnitude of the smooth muscle response.

Segments of small intestine of some, but not all, guinea pigs react to the presence of rabbit anti-γ globulin. The differences that have been encountered may be due to genetic variation in guinea-pig γ globulins.

Previous findings have been confirmed that normal γ globulins may interfere with the sensitization of tissue or with the tissue response due to specific antibody.

In vitro sensitization of guinea-pig small intestine demonstrated that the smooth muscle response to one immune system may diminish a subsequent response of the same tissue to an unrelated antigen-antibody reaction.

     

The sheep immune response: Variation of anti-hapten and anti-carrier antibodies localized in the γ1 and γ2 immunoglobulin fractions

The results obtained in the quantification of γ₁ and γ₂ anti-hapten (DNP) and anti-carrier (HGG) antibodies obtained weekly during 50 weeks and proceeding from a group of three Merino sheep immunized with DNP—HGG, are described.

The primary and secondary response and the antibody variations in weekly repeated stimulations were studied.

The anti-DNP and anti-HGG total antibodies have similar variations. γ₁ and γ₂ anti-DNP and anti-HGG antibodies do not show differences in the primary response nor in weekly repeated stimulations.

In the secondary response, a high increase of γ₁ with loss of γ₂ is observed. After a certain time γ₁ decreases and γ₂ increases.

     

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 antibody activities of 19S and 7S fractions from rabbit antisera to Bordetella pertussis

Bordetella pertussis antisera were fractionated on Sephadex G-200 to give 19S and 7S globulins, the latter refractionated on DEAE-cellulose, and the antibody properties of the two purified materials compared.

The minimal weights of 19S and 7S globulins required to neutralize haemagglutinin and to protect mice against an intranasal challenge causing lung infection were similar. In agglutination 7S globulin was 1.5–100 times more effective than 19S, the ratio depending on strain and serum, but there was no apparent correlation with specific agglutinogens.

7S globulin was at least 100 times more effective than 19S both in the complement-dependent bactericidal reaction in vitro and in the protection of mice against an intracerebral challenge. It is suggested that the two reactions measure the same antibody.