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An indirect immunofluorescence test using small discs (0.4 cm in diameter) cut from blood smears on filter paper is described. Discs plus three drops of phosphate-buffered saline are laid on antigenic areas on slides, which are then incubated. During incubation, eluted antibodies react with antigen. The method was tested with 10 eluates from healthy persons and with 30 eluates from three groups of 10 patients with Chagas’ disease, schistosomiasis Mansoni and South American blastomycosis, respectively. Sera from the same blood samples were tested also by conventional indirect immunofluorescence tests. Both tests gave similar results. The method proposed is useful as a rapid qualitative screening test when dealing with a large number of samples.

The principle and procedure of two-dimensional crossing paper electrophoresis are described. In applying the technique to the antigen-antibody reaction, electrophoresis of the antiserum alone was carried out in one direction, then the antigen was applied on a line to encounter each fraction of the antiserum and electrophoresis was started in a direction at right angles to the first one. Peaks appeared on the line of antigen owing to the formation of complexes in the regions of antibody proteins contained in the antiserum, thus forming a “crossing diagram”. An anomaly was observed in several cases of the “crossing diagrams” of egg albumin-anti-egg albumin reaction; it was ascribed to mechanical disturbances of the paper surface. A “crossing diagram” of a diphtheria toxoid obtained by initial electrophoresis of the antiserum showed two peaks in the region of T-globulin and one in the region of γ-globulin of the antiserum. Reversing the order of electrophoresis (first toxoid, then antiserum) the “crossing diagram” showed two flat asymmetrical peaks in the γ-globulin zone. In general, the toxoid used must contain more than four antigens, of which two have their antibodies in T-globulin and the other two in γ-globulin. It is suggested that the antibodies have different equivalence zones for the same toxoid.

AGAR diffusion1 has been used for detecting antigens after paper electrophoresis. I have found that a precipitin reaction can be carried out directly on the paper.

Paper disc-electroimmunodiffusion in agarose gel provides a rapid and sensitive quantitation of the immunoprecipitin reaction.

SummaryA method for the chemical identification of histamine in blood has been described and used to prove the appearance of histamine in plasma after addition of antigen (in vitro) to the blood of sensitized rabbits and in the blood of dogs undergoing anaphylactic shock.

Immunoelectrodiffusion with paper discs is a simple and rapid method for screening blood donors for hepatitis antigen.

Reaction of flocculation between the antitoxin ofCl,oedematiens and the corresponding immune horse serum was studied with the aid of precipitation on paper. When toxin and immune serum were placed on the same place on the paper, a flocculate appears, which unlike all the rest of the proteins is not washed off the paper by the veronal buffer. The fiocculate is precipitated at the zone of considerabic surplus of antitoxin. The quantity of precipitate is increased in proportion to the quantity of the added antigen up to the time when the whole quantity of the antigen is precipitated. Then the precipitate begins to dissolve in the surplus of the antigen. Data which were obtained by the method of precipitation on paper correspond to the results of determination of the quantity of the antibodies in the same serum with the aid of the antigen fixed on paper through diazoconnection and the residual haloidalkylate. This points to the possibillity of replacement of the biological method of determination of the antigens ofCl.oedematiens by the immunochemical method.

A specific addition of protein nitrogen was obtained in the agglutinate of dermatol particles, covered by antigen. Protein combined with azo-dye with bis-diazotized dianisidine gives stable sorption on the surface of the paper, cotton material or cotton wool. Such paper azo-antigen gives a pronounced specific addition of protein nitrogen which comes from the proteins of the antiserum. Analogous data were obtained with native paper antigens, secured by simple drying of the protein solution in which chromatographic paper is soaked. Reaction of specific fixation of the protein nitrogen on the surface of the paper antigen represents an easily performed new serological reaction in immunology. This reaction gives a numerical value of quantitative characteristics of the immunological antigen-antibody relation.

Using a standardized procedure of paper electrophoresis, seven fractions were consistently separated from parotid saliva. These components were eluted from the paper strips and analysed chemically and immunochemically. Serum proteins (albumin, α, β and γ-globulins) comprised 25 per cent of the total. The specific salivary proteins included amylase, two non-antigenic glycoproteins, and an unidentified antigenic cationic protein. The two glycoproteins (accounting for 35 per cent of the total protein) were cationic, contained hexoses, fucose and hexosamine in a ratio of 1:1 and little if any sialic acid.

Protein antigens fixed on paper in the form of azocompounds or by simple drying at room temperature have a high serological activity and specifically fix the complement in the presence of a corresponding anti-serum. This was established by the method of quantitative reaction of complement fixation (50% titer). Complement fixation test (50% titer) for “paper” antigens gives an opportunity of using different antigens in the same test and replacing them during the experiment. This permits the use of antigens adsorbed on paper and the quantitative complement fixation test for investigation of the optimal conditions of obtaining the specific antisera.

The IgG M-components of 70 sera were quantitated by a paper electrophoretic method and by an immunochemical method with the use of an antiserum raised against purified polyclonal IgG. The results obtained by the two procedures were concordant and thereby showed that IgG M-components of different antigenic compositions can probably be quantitated by simple immunochemieal methods. Immunochemical determination of the total IgG content of sera containing IgG M-components by the Mancini technique agreed, both when the sera were assessed in their native and in their carbamylated form, with the immunochemical quantitation of Weeke. This indicates that different IgG M-components are immunochemically similarly altered by carbamylation.

Immunoelectrophoretic analysis of an egg yolk livetin preparation has demonstrated the presence of six distinct fractions, which were numbered 1, 2, 3, 4, 5, and 6 from the anodic end of the electropherogram. Four of these fractions have been identified with four livetin fractions obtained by paper electrophoresis as follows: immunoelectrophoretic fraction 1 with alpha-livetin, fraction 2 with beta-livetin, fraction 4 with gamma1-livetin, and fraction 5 with gamma2-livetin.Immunoelectrophoresis of hen serum or cock serum against antilivetin serum has shown that both hen and cock sera contain antigens electrophoretically and immunologically identical to alpha-, beta-, gamma1-, and gamma2-livetins.

Thyroglobulin was prepared from human thyroid glands and labeled in vitro with I131. Zone electrophoresis (pH 8.6) of mixtures of serum with labeled thyroglobulin revealed that the anodal mobility of thyroglobulin was decreased appreciably in sera rich in thyroglobulin auto-antibody (hemagglutination technique) as compared with serologically negative specimens. The results of experiments in which the ratio of antigen to antibody was varied suggested that the decreased electrophoretic mobility in antibody-containing sera might be ascribed to the formation of thyroglobulin-antibody complexes of mobility intermediate between that of gamma globulin and of thyroglobulin. Paper electrophoresis of serum containing labeled thyroglobulin may be useful for detecting thyroglobulin auto-antibody, although the procedure is less sensitive than the tanned erythrocyte agglutination method.

Some Wassermann-positive serums give precipitation reaction on paper with diluted lipoid antigen. Preliminary drying of serum on paper and employment of nondiluted antigen in certain ratios considerably increased the sensitivity of this method. It is presumed that Wassermann reagents are precipitated on paper with lipoid antigen.

Publisher Summary This chapter elaborates the potentials for immunologic intervention in cancer. Several findings in tumor immunology strongly suggest that practical methods will be forthcoming to permit the detection and measurement of tumor antigens in human cancer patients. Tumor-specific immunity, both cell mediated and that based upon humoral antibodies can be assessed in vitro on an experimental basis. With the exception of the fluorescent antibody techniques and perhaps, one or two complement fixation methods, all of these tests depend upon tissue culture-derived tumor specimens. The in vitro management of such cells mixed with antibody, putatively immune cells or both, involves techniques which are difficult from the technical point of view and based upon the assessment of effects which are rarely all or none. It has been emphasized that the central problem in tumor immunotherapy is not that of inducing the patient to become immune to products of his tumor but rather to make existing, demonstrable immunity more effective in controlling tumor growth.

The growth inhibition test for identifying Mycoplasma species has been modified by drying antibody-impregnated paper discs at 5 C. When stored at −20 C, these discs have been found to retain their inhibitory activity for longer than 7 months. Since these discs can be stored for long period of time, significant advantages over present methods result. When, for example, discs are arranged on a ring, a single test can be used for the identification of an unknown human species. Valuable antisera can be distributed to other laboratories on paper discs in much less volume than can fluid antiserum. Considerable savings of time result from prior preparation of many discs that can then be stored and used over a long period of time. The growth-inhibiting antibody is stable, and the activity is not enhanced by a heat-labile accessory factor from fresh guinea pig serum which increases the antibody titer in the metabolic inhibition test.

Publisher Summary This chapter discusses the fundamentals of immunogenetics and their application to histocompatibility. This chapter focuses on the basic principles underlying the genetic control of transplantation antigens and on the methods used both to detect these antigens and to analyze the data obtained with the various methods. Antigens are histocompatibility antigens if they can be related to graft rejection. In the absence of treatment protocols designed to delay or prevent graft rejection, any histocompatibility antigen will lead to graft rejection if the antigen is present on donor tissue and absent from the host. Some histocompatibility differences are probably present in all donor-recipient combinations, with the exception of identical twins in man and inbred strains in other animal species.

Publisher Summary This chapter focuses on lymphocyte response as a model of cell-mediated immunity. The initial response (recognition) by lymphocytes to either antigens to which the donor of the cells is sensitized or to allogeneic cells involves, at least in part, different cell populations responding to the different stimuli. Likewise, at least partial specificity was demonstrated by cells sensitized in vitro in the mixed leukocyte culture reaction in terms of an in vitro cytotoxic reaction mediated by such cells. On the other hand, the adherent cell requirement for the MLC reaction appeared to be nonspecific, as were a variety of soluble “factors” which could influence lymphocyte response. Lymphocytes from animals primed with certain antigens undergo blast transformation and mitosis in vitro when cultured with the priming antigen. It was demonstrated that different populations of antigen-specific precursor cells exist in the spleens of immunized mice. It was also observed that addition of cell-lethal amounts of tritiated thymidine to cultured cells dividing in response to sheep erythrocytes left a cell population which could no longer produce plaque-forming cells to the homologous antigen but would produce plaque-forming cells in response to a heterologous non-cross-reacting antigen.

Publisher Summary This chapter discusses leaf dip serology. Without elaborate purification, a virus can be classified on the basis of gross morphology by dipping the infected leaf in a drop of water. The leaf-dip procedure is modified by incorporating virus-specific antiserum into the drop and making the initial observation of the virus, a positive method of identification. Antibody-covered antigen when negatively stained has a thick, dark appearance, making labeling of the antibody with electron- dense material unnecessary. By this procedure, plant viruses can be easily distinguished even when mixed with morphologically similar particles. The advantages of the test are that: (1) the antiserum sample can be stored conveniently for immediate use; (2) it requires very small amounts of antiserum; (3) the reaction of the virus rod and antibodies can be observed directly; and (4) the occurrence of other morphologically similar viruses in the test does not obscure the test or give false positive results.

Heparin precipitates of human and rat serum lipoproteins, dissolved, dialyzed, and analyzed by paper electrophoresis, were found to migrate as β-lipoproteins. The pre-β-lipoproteins disappeared. Using specific anti-human β- and α-lipoprotein sera it was shown that the heparin precipitate of human very low density lipoproteins (VLDL) contained β-lipoproteins, whereas the supernatant contained α-lipoproteins. This indicates that these components are dissociated by the heparin precipitation procedure. Following partial delipidation of the heparin precipitate, a procedure which exposes the antigenic site of the α-lipoprotein of the VLDL, it can be demonstrated that the α-lipoprotein component is completely removed by the heparin precipitation procedure. Partially delipidated rat VLDL showed four immuno-reactive components when reacted against rabbit anti-rat VLDL serum, but only one, corresponding to the β-lipoprotein, when reacted with rabbit antiserum to heparin precipitate.Chemical analysis of the lipid components of the heparin precipitate and supernatant of human and rat VLDL reveals that over 90% of the triglycerides are precipitated by the heparin and that the supernatant fraction contains lipoproteins having a phospholipid to protein ratio higher than any of the other lipoproteins.Upon reincubation of the dissolved and dialyzed human serum heparin precipitate and its supernatant a pre-β band reappeared on paper electrophoresis. The recombined pre-β-lipoprotein was isolated and found to react with anti-β-, but not with anti-α-lipoprotein serum. This is a characteristic of native pre-β-lipoproteins. Evidence has also been obtained that the high density lipoproteins combine only with the β-lipoproteins originally present in the VLDL.

SummaryA new method of demonstrating the union of antigen and antibody is described. The test is performed'on the surface of filter paper and submitted to the washing effect of a stream of isotonic NaC1 solution. While normal serum is washed together with the antigen, the immune serum produces the fixation of the antigen to the paper at the place where it was deposited.Regarding its specificity, it has been observed that this test, studied only in brucellosis, produces responses which so far have agreed with the usual agglutination reactions.

SummaryThe nature of phosphatide antigens of 5 species of mycobacteria has been investigated by the kaolin-agglutination technique of Takahashi using serum from tuberculous patients as antibody source. The phosphatide antigens are present in virulent (H37 Rv, M. avium), avirulent (H37 Ra) and saprophyte (M. 607, M. phlei) strains of mycobacteria. The phosphatide antigens have been purified by silicic acid column and paper chromatography and identified as phosphatidyl inositomannosides.

Antigenic extracts of Streptococcus group E (SGE) were subjected to fractional ethanol precipitation, block (preparative) electrophoresis, and gel filtration for the purpose of separating the type antigen from the group antigen. Ethanol precipitation was ineffective in separating the substances. Block electrophoresis yielded serologically pure group antigen and a mixture of type and group antigen. Serologically pure type antigen was obtained by gel filtration. In some cases, pure group antigen was also recovered; in others, it was contaminated with type antigen. Gel filtration column effluents of antigenic extracts of SGE serotypes, I, II, III, IV, V and “untypable” isolates, collected from the region in which type antigen was eluted, were studied by paper chromatography and infrared spectrophotometry in an effort to develop a nonserological means of detecting type antigen. Hydrolysates of type antigens or suspect type antigens of serotypes I through V contained l -rhamnose, d -glucose, and several unidentified substances. d -Galactose also was present in hydrolysates of serotypes III and V. Untypable isolates and negative controls contained traces of d -glucose only. The data suggested that serotypes I through V contained a type antigen and that the untypable isolates were devoid of type antigen. Infrared absorbance spectra seemed to support the paper chromatography data.

The only aspects of the genetics of immunoglobulins which will be discussed in this short paper are those which are directly related to the antigenic specificities carried by these proteins, and these have been discussed recently in a more detailed paper (Oudin 1966). Three kinds of antigenic specificities with different genetic meanings may be distinguished and will be considered in immunoglobulins. They may be discussed also in terms of antigenic patterns, that is, in terms of the structures still unknown which are responsible for these antigenic specificities or, in other words, for the combination of the immunoglobulin molecules with antibody molecules; each antigenic pattern is made of an unknown number of specific groups or determinants which may combine with as many antibody molecules. The isotypic specificities are those which are uniform in all individuals of one animal species (Oudin 1960 b ; Dray et al . 1962). Since they were the only kind of antigenic specificity known before 1956, they did not deserve at that time a special denomination. These isotypic specificities make it possible to distinguish several main classes, now termed IgGr, IgM, IgA among the immunoglobulins, in a manner which is probably more convenient and precise than the usage of physico-chemical characters. The isotypic specificities also permit one to distintinguish, within one class, subclasses which are found in all normal individuals of a species. If two immunoglobulin molecules are endowed with somewhat different isotypic specificities, this would seem to imply that the synthesis of these molecules, or rather of at least one of the polypeptide chains which they contain, is controlled by different structural genes.

A human macroglobulin (γM) was separated from the serum of normal donors who had been immunized with typhoid vaccine. The γM antibody to Salmonella typhi O 12 antigen was obtained by chromatography of the γM-globulin on a column of specific immunoadsorbent. The somatic antigen, i.e. the lipopolysaccharide of S. typhi, was extracted from the bacterium and partially hydrolyzed. A tetrasaccharide, which is the antigenic determinant of the lipopolysaccharide, was separated by paper chromatography. Equilibrium dialysis of the purified γM antibody with the tetrasaccharide showed that the antibody had 10 antigen-combining sites per molecule and an association constant with antigen of 2.7 x 104.

Bolaños, Roger (Tulane University, New Orleans, La.), and Charles W. DeWitt . Isolation and characterization of the K1 (L) antigen of Escherichia coli . J. Bacteriol. 91: 987–996. 1966.—An acidic polysaccharide with the serological properties of the K1 (L) antigen has been isolated from Escherichia coli O2:K1:H4 by means of phenol-water extraction, fractionation with hexadecyltrimethylammonium bromide (Cetavlon), filtration through Sephadex G-200, and chromatography on anion-exchange cellulose columns. Nucleic acid and protein content were reduced to a nondetectable level. There is no contamination with O antigen. The active material appears in two different positions in the Cetavlon fractionation, each with a slightly different serological specificity, as followed by the inhibition of passive hemagglutination. By paper chromatography, the polysaccharide moiety of the O antigenic fraction is composed of glucose, galactose, hexosamine and rhamnose. The absence of colanic acid in either K1 fraction was not proven, although its participation in our assay system, as well as the participation of the common or cross-reacting antigen, was ruled out by serological means.

SUMMARY A fraction (P. 1/2/3 ) precipitated from urine with 3 volumes of alcohol, contained approximately 5·6% Kjeldahl nitrogen, 42·4% carbohydrate (estimated as galactose), 11·9% hexosamiue, but no nucleic acid. One of the agar gel precipitin bands was shown to contain lipid. By the use of paper chromatography on the hydrolysed fractions, galactose was demonstrated, and possibly sorbose and arabinose, together with some amino acids. The antigens were resistant to a temperature of 94° C. for 60 min. and to the action of trypsin. Separation of the individual antigens was not obtained by either ultra-centrifugation or electrophoresis. Fraction P. 1/2/3 fixed complement in the presence of hyperimmune sheep sera, and absorbed 87·5 and 97% of the agglutinating antibodies and 94 and 87·5% of the complement fixing antibodies respectively from 2 hyperimmune sheep sera. This fraction was weakly pyrogenic in rabbits and non toxic to cattle, rabbits and mice, but proved to be lethal to chicken embryos. The antigens behaved as haptens in cattle, rabbits and mice, but they produced precipitating antibodies in rabbits when they were combined with ‘shiga conjugated protein’. Fraction P. 1/2/3 possessed an aggressive action when inoculated with viable Mycoplasma mycoides in immune animals and appeared to enhance the virulence of the organisms in susceptible cattle. It did not produce a delayed allergic reaction in immune cattle. It is suggested that these antigens play a part in assisting the growth of M. mycoides in the host tissues but are not in themselves significantly harmful.

An adequate amount of typhoid-paratyphoid vaccine (TPV) or bovine liver lipid which had been emulsified in bovine serum was injected intravenously to rabbits of two groups two times at an interval of 4 days. After the second sensitization, it was found that a new specific substance had appeared in the circulating blood of each rabbit, and that it had accelerated the endogenous respiration of mouse liver cells remarkably under aerobic conditions.An effective serum was used as a starting material and examined first for stability against heat, acid, and alkali and dialyzability agains semi-dialytic cellophane membrane. Then, the extraction and purification were carried out by the column-chromatographic method. In this method, the deproteinized serum filtrate was absorbed on Amberlite IR 120H+ resin with 100-200 meshes. After that, the effective substance was extracted as a single substance through the column-chromatographic fractionation by effusion of 0.1N-HCl solution. Finally, it was identified as uric acid by paper chromatography, continuous ultraviolet and infrared absorption spectra, element dialysis, and measurement of melting point. On the other hand, uric acid was compared with some metabolic intermediates of ATP, with the exception of uric acid itself, as substrates for the oxidative respiration of mouse liver homogenate or mitochondria.

A patient who developed thrombocytopenia while taking analgesics was found to have circulating antibody to a metabolic product of N-acetyl-p-aminophenol (NAPA), but not to the drug itself. The antibody reacted in vitro with an antigen present in the blood of a normal volunteer who had ingested NAPA. The antigenic material was purified from the urine of this volunteer and shown to be the sulfate conjugate of the drug. These findings demonstrate that a drug metabolite may be antigenically active in drug-induced thrombocytopenia when the drug itself is immunologically inert, representing a form of drug-induced thrombocytopenia.

THE antigenic properties of insulin are now well recognized, but no simple method has yet been described for the routine detection and assay of insulin antibodies. The method described here is based on the classical observation by Berson, Yalow, Bauman, Rothschild and Newerly1. They showed that during chromato-electrophoresis on filter paper, 131I-labelled insulin bound by insulin antibodies in human serum moves with the β-γ-globulins, while free insulin remains fixed at the point of application of the serum-insulin mixture on the filter paper. When an excess of unlabelled insulin mixed with a trace of labelled hormone is added to the anti-insulin serum, the excess insulin is adsorbed from solution by cellulose and the amount bound by the antibodies is measured in the supernatant solution. We have used this assay system for serum obtained from guinea-pigs treated with bovine insulin2 and capable of neutralizing insulin at rates ranging from 0.1 to 3.5 units per ml. serum. It can, however, be adapted for the detection and assay of antibody concentrations lower than this; it has not yet been used for anti-insulin sera of other animals or of man.

A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.

Twenty-seven Bence Jones proteins of immunological type K show a common set of peptides. One 6f the commnon peptides differs in three of the proteins which are the only ones classified by a serological test as Iav(a+). The difference in the peptide analyzed is caused by a valine-leucine interchange; Inv(a+) proteins have leucine, whereas Inv(a-) proteins have valine in position 189.

An allergenically active basic protein, antigen Ra5, was isolated from DEAE-fraction C of the aqueous extract of short ragweed pollen by gel filtration and cation exchange chromatography. The antigen was homogeneous by a number of physicochemical and immunological criteria, and showed no cross-reaction with ragweed pollen antigens E, Ra3 and BPA-R. It was devoid of carbohydrate, had a molecular weight of 5,130 ± 50 in the analytical ultracentrifuge and the following composition: lys4 arg2 asp2 ser4 glu4 pro3 gly4 ala3 (cys/2)6 val3–4 ileu leu1–2 tyr3 try2.

In order to determine the structure of the tumor-specific immunodominant group of the carcinoembryonic antigen (CEA) of human gastro-intestinal tumors, immunologically active fragments from this glycoprotein were prepared by partial enzymatic hydrolysis using the proteolytic enzyme nagase. The fragments were purified by sequential molecular sieve, adsorption and ion exchange chromatography, and were analysed for carbohydrate and amino-acid composition by gas-liquid chromatography. The antigenic activity of each fragment was tested using both the Z-gel and Farr radioimmunoassay techniques for CEA. Immunologically active glycopeptides with molecular weights of 1,000–5,000 Daltons, which contained the tumor antigenic determinant of CEA, had relatively high contents of N-acetyl-D-glucosamine, aspartic acid or asparagine, and glutamic acid or glutamine.

A purification procedure applied to H-2 antigens (controlled by the H-2 locus) was used for the characterization of HL-A substances (HL-A2 and HL-A7) (controlled by the HL-A locus) solubilized from lymphoid cell membranes by delipidation. Upon Sephadex chromatography the serologically active material had an apparent Mr of 32000, and displayed, in relation to H-2 antigens, a faster electrophoretic mobility and higher pI values. One sulfhydryl group and one (intrachain) disulfide bond were present in partially purified HL-A preparations. Reduction and aminoethylation in 4 M urea did not influence the electrophoretic migration of HL-A active substances, suggesting that, as already demonstrated for H-2 alloantigens, in HL-A molecules the genetic determinants are present on single polypeptide chains. Highly purified HL-A alloantigens, like H-2 substances, revealed a high degree of microheterogeneity. These findings further support the concept of the genetic and structural homology of the main histocompatibility systems in mouse and man.

Serum from a patient with multiple myeloma exhibited an unusual concurrence of abnormalities: (1) bitypic gammopathy consisting of a whole Λ-IgG 1 paraprotein plus a circulating Λ-Bence Jones protein; (2) marked insolubility of the partially purified paraprotein under conditions of low ionic strength, but good solubility upon further purification; (3) release of an unusual low molecular weight fragment containing Fc antigenic determinants under standard conditions of mercaptoethanol reduction of the purified paraprotein. Evidence is presented supporting the origin of the Fc fragment from the paraprotein heavy chain and describing the requirement for 6 M urea for its dissociation from the reduced and alkylated heavy chain. The phenomenon is evidently not rare, since screening of 14 additional myeloma sera revealed one with similar euglobulin properties and an apparently similar Fc fragment abnormality. The nature and origin of the fragment in relation to previously described immunoglobulin fragments are discussed.

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient's blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.

A patient is described who developed renal failure due to a severe proliferative glomerulonephritis with hypocomplementaemia and cryoglobulinaemia while repeatedly injecting herself with diphtheria, pertussis, and tetanus vaccine (D.P.T. vaccine). After stopping the antigen administration there was recovery of renal function, complement values returned to normal, and cryoglobulin could no longer be recovered from the sera. The pathogenesis of the glomerulonephritis is discussed.

O antigen mutants were obtained from Salmonella durban , a group D 1 organism, by treatment with N -methyl- N′ -nitro- N -nitrosoguanidine. Serological studies demonstrated that the mutants lost the O-9 antigen factor of the parent organism but acquired the O-2 factor specific to group A Salmonella . Lipopolysaccharides of the mutant strains contained paratose which determines the specificity of O-2 factor. Tyvelose, present in the wild-type lipopolysaccharide, was not found in the mutants. H antigens and other biological characteristics of the mutant strains were the same as those of the wild-type organism. The present finding implies that group A Salmonella species might be derived from group D 1 organisms.