Isolation and characterization of an asthma-inducing sea-squirt antigen

An antigen capable of inducing asthmatic attack in patients with sea-squirt allergy has been isolated from body fluid of sea-squirt, Styela plicata, and designated as DIIIa. The chemical composition and the behavior in anion-exchange chromatography showed that the antigen was a weakly acidic glycoprotein. The weight-average molecular weight of DIIIa was estimated to be 9,880 by the sedimentation equilibrium method. This antigen was apparently discriminated from the previously isolated sea-squirt antigens, Gi-rep and Ei-M, by its activity to induce asthmatic attack and conjunctival congestion, though it gave a strongly positive reaction in skin tests against allergic patients, similarly to the previous antigens. From the cross-reaction of DIIIa to rabbit anti-Gi-rep and anti-Ei-M sera in vitro, it was confirmed that the antigen carried essentially the same antigenic determinant as Gi-rep and Ei-M, and this was termed type alpha determinant. Furthermore, the allergenic activities detectable in vivo and the reactivity to the rabbit antisera in vitro were simultaneously absorbed by immobilized immunoglobulin from anti-Gi-rep serum. Thus, it was suggested that the antigenic determinant responsible for the allergic reactions in vivo corresponded to that specified as type alpha in vitro on the basis of the reactivity against the rabbit anti-Gi-rep serum.     

应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照,对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析;结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM),鉴定了特征色谱峰;考察了方法的重复性和检测线性。结果表明,两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离,在保留时间约13.3 min处出现抗原特征峰;MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰,分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da,接近PCV2的理论分子量;同时在保留时间约11.4 min处也出现色谱峰,经检测分子量为4.37×10⁶(±0.42%) Da,TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测,抗原色谱峰面积的RSD(n=3)均小于1.5%,重复性好;将PCV2 VLP纯品梯度稀释检测,VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系,R²分别为0.999及0.997,能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法,用于质量评价与提升。     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

小鼠颌下腺表皮生长因子的分离、纯化与鉴定

雄性成年小鼠颌下腺醋酸提取物经Bio-Gel p-10凝胶层析可得七个色谱峰,经放射受体结合及放射免疫分析后,证实第七峰的EGF比活性最高。收集该组分,再经Sephadex G-10层析及DEAE-52离子交换层析,可得纯化的EGF样品。SDS-PAGE证明样品的分子量为6 000。RRA,RIA结果表明,样品与标准mEGF具有相同的受体结合活性及抗原性。生物学研究证明样品可以促进新生鼠睁眼和萌牙,刺激成纤维细胞DNA的生物合成。     

Purification and characterization of a sea squirt beta-galactosidase

A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.     

The type antigen III of group F streptococci separation of group and type antigens and partial characterization of type III antigen

Polysaccharides extracted from Z₃III streptococci either with formamide or with dilute hydrochloric acid or isolated from the growth medium could be fractionated in type III- and group Z₃ antigens by alcohol precipitation. No good separation could be obtained from TCA extracts. When the same extractions and fractionations were applied to streptococci carrying type III antigen, but different group antigens, good separations were again obtained of all formamide extracts, but not of all hydrochloric acid extracts. The group antigens showed a rhamnose content of at least 50% and contained hexosamines. Type III antigens contained mainly rhamnose, glucose and galactose in relative amounts of approximately 1:2:3. Analysis of the methylated type III antigen suggests it to be a polysaccharide with a linear structure. Type III antigen isolated from the medium was characterized not only by a different sugar ratio, but also by its fucose content of 20%. In some cases the purified polysaccharides contained considerable amounts of glycogen-like material. Partial acid hydrolysis of the type III antigen extracted with formamide yielded a great number of oligosaccharides. Analyses, inhibition reactions and methylation studies gave indications that the most probable structure of a determinant group of type III antigen is β-glucosyl-(1-6)-galactosyl-(1-6)-galactosyl-(1-3)-rhamnose. The possibility of the existence of a second determinant group is discussed.     

Isolation and dissociation of immune complexes from pleural effusions of lymphoma patients

Summary Immune complexes (IC) isolated from pleural effusions of lymphomas with favorable and unfavorable prognoses were of IgG type. These IC were further dissociated by ion exchange chromatography using 8 M urea. The antibody was found to be a high molecular weight protein (1.5×10⁵ daltons) and reacted with antihuman IgG immunologically while a second peak obtained on ion exchange chromatography may be an antigen moiety with a molecular weight of 3.2×10⁴ daltons as it reacted immunologically with the antibody. Strong cytoplasmic fluorescence was observed with various cell suspensions of lymphomas when reacted with the antibody preparations. The antisera raised against two different antigen fractions prepared from two lymphomas — nHL and LL showed positive fluorescence with both nHL and LL suspensions. The absorption of these rabbit antibodies with individual cell extracts or with antigen preparations also entirely blocked the cytoplasmic staining. The antigen moiety (PK-II) may have a common origin in the disease process. Pleural effusions from patients with unfavorable and favorable prognoses showed identical patterns of separation of IC components.     

Purification and properties of antibodies having specificity for the carbohydrate moieties of synthetic glycoconjugates

Three types of antibodies having specificity for carbohydrate moieties of glycoconjugate antigens have been isolated from the sera of rabbits immunized with vaccines of β-d-glucosyl-bovine serum albumin, β-d-galactosyl-bovine serum albumin, or β-lactosyl-bovine serum albumin. The antibodies were isolated by affinity chromatography on adsorbents bearing appropriate carbohydrate ligands. The antibody types are specific for the β-d-glucosyl, β-d-galactosyl, or β-lactosyl groups of the synthetic glycoconjugates. The specificities have been established by data on hapten-inhibition, agar-diffusion, periodate-oxidation, and affinity-chromatography experiments. Each antibody type is of the IgG class of immunoglobulins and is of uniform molecular size, with molecular weight of 1.5 × 10⁵. Data from gel-isoelectrofocusing experiments showed that each preparation of antibodies, although specific for identical determinant groups of the same antigen, nevertheless consisted of a set of different proteins. The anti-d-glucose antibodies consisted of five proteins, the anti-d-galactose antibodies of eleven proteins, and the anti-lactose antibodies of seventeen proteins. The suggestion has been made earlier that the members of such a set of antibodies, in which each member is induced by the same determinant group of an antigen and each member combines with this group in the precipitin reaction, be called isoantibodies.     

Immunochemical detection and characterization of pregnancy-associated endometrial alpha 1- and alpha 2-globulins secreted by human endometrium and decidua

Antisera raised against the soluble antigens of the endometrium of early pregnancy detected two antigenic proteins of alpha 1 and alpha 2 mobility in extracts of this tissue and were termed antigens A and B. Neither antigen was detected in pregnancy sera or extracts of proliferative endometrium, but antigen B was detected in extracts of secretory endometrium and both were present in amniotic fluid and medium from in-vitro incubations of pregnancy endometrium. Fractionation of radiolabelled medium on ion-exchange chromatography demonstrated that antigens A and B co-eluted with the proteins from which EP14 and EP15 were derived and which were the major secretory polypeptides of pregnancy endometrium in vitro. Further biochemical purification revealed that EP14 (Mr 32 000) was derived from a protein of native molecular weight 36 000 which existed in two forms, whereas EP15 (Mr 28 000) was derived from a dimeric glycoprotein of native molecular weight 56 000. Immunochemical studies demonstrated that antigens A and B are identical to these two secretory proteins and have been termed pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG).     

Evidence for a high molecular weight cytosolic factor that binds brain and liver metallothionein

When brain extracts were fractionated in a Sephadex G-75 chromatography and MT levels were assayed by RIA or ELISA using polyclonal antibodies specific for the MT-I and MT-II isoforms, it was found that MT mostly eluted in the high molecular weight (HMW) peak even in reducing or anaerobic conditions. This was also the case for the liver extracts of control rats; in stressed animals MT immunoreactivity in the HMW peak (>80 Kd) was increased compared with undisturbed animals, but the major amount of the newly induced MT eluted, as expected from the current literature, in the low molecular weight (LMW) peak, around 10 Kd. The addition of purified MT to brain extracts precluded its binding to a DEAE-Sephadex column. Furthermore, immunoblot results of native PAGE showed that MT changed its electrophoretic mobility in the presence of HMW proteins from brain cytosol. Altogether, these results suggest that a cytosolic factor binds MT in a saturable manner, which may have strong physiological implications.     

Antibody responses to antigens of Streptococcus mutans in monkeys (Macaca fascicularis) immunized against dental caries

Immunization of rabbits or monkeys with walls prepared from Streptococcus mutans by a procedure including extraction with SDS at room-temperature induced antibodies to three antigens (A, B and C) detectable by crossed immunoelectrophoresis. Antigens A and B have previously been characterized as proteins of molecular weight 29 000 and 190 000, respectively. Antigen C was characterized as having a molecular weight of 70 000 and was purified by immunosorbent affinity chromatography and hydrophobic interaction chromatography. Another wall protein, antigen D, of molecular weight 13 000, was extracted from walls with Triton X-100. Immunization of monkeys with walls prepared from cultures of S. mutans grown at a high (D = 0.5 h-1) or low (D = 0.05 h-1) dilution rate in a chemostat showed that only the latter induced protection against dental caries. There was a positive correlation between levels of antibody to antigens A and C and induction of protection and a negative correlation between protection and the level of antibody to antigen B. No antibody to antigen D was detected in protected monkeys and an experiment in which monkeys were immunized with pure antigen D confirmed that it does not induce protection.     

Grouping antigens of four Lactobacillus species and their characteristics.

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2: 1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4: 1. Inhibition tests indicated that the immunodominant component of antigen 9 was a-methylglucoside (glucose), and most probably the determinant is a glycosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Comparison of Solid-Phase Radioimmunoassays for Baculoviruses

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.     

Schistosoma mansoni: Use of antigens from excretions and secretions in immunodiagnosis

The use of antigens from excretions and secretions (ESA) of Schistosoma mansoni in two immunodiagnostic tests, the enzyme-linked immunosorbent assay (ELISA), and the defined antigen substrate spheres (DASS) system, has been extensively investigated. In comparison with total adult worm antigens (AWA), the sensitivity of the DASS tests remained the same, while that of the ELISA increased slightly when ESA was used. For further analysis, the ESA preparation was fractionated according to molecular weight, by gel filtration. The humoral immune response of immunized rabbits, infected mice, and humans to each of these molecular-weight fractions was determined by incubating an equal, nonsaturating amount of each ESA fraction in a double-antibody sandwich system, using Sepharose beads as a carrier. The humoral immune response of rabbits immunized with ESA was primarily directed against antigens with molecular weight between 50,000 and 70,000. In contrast, immunoglobulins from sera of infected mice or humans, reacted well with antigens from a large molecular-weight range. Screening of a large number of sera for the presence of specific antibodies is most conveniently executed with tests in which antigens, instead of antibodies, are bound to a matrix. However, binding of antigens to Sepharose beads or polystyrene microtiter plates was shown to decrease considerably with decreasing molecular weight of the antigen. Therefore, of all ESA fractions, those containing the high-molecular-weight antigens (MW > 200,000) gave the most sensitive DASS and ELISA tests. These high-molecular-weight excretory and secretory antigens, in contrast to a total-worm homogenate, and excretory and secretory antigens with a molecular weight lower than 200,000, possessed a high specificity for S. mansoni. The specificity of the high-molecular-weight preparation was shown to be mainly due to the presence of the circulating anodic polysaccharide antigen, since removal of this antigen by immunoadsorption led to a considerable decrease in specificity.     

Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.     

Purification and Properties of a Thermo-labile Antigen from Baker’s Yeast Cells

A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5 x l0⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, N-acetylglucosamine and fucose. The antigenic determinant of TLA was distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLA was heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLA agglutinated fresh yeast cells, and the distribution of TLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLA molecules were exposed on the outer surface of the yeast cell wall.     

Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics

Cohen, Gary H. (University of Pennsylvania, Philadelphia), and Wesley C. Wilcox. Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics. J. Bacteriol. 92:676-686. 1966-Infection of mammalian cells with members of the poxvirus group elicits production of a number of virus-induced, soluble antigens. Immunoelectrophoresis and immunodiffusion techniques employing soluble antigen preparations obtained from vaccinia virus-infected KB cells revealed at least seven well-defined immunoprecipitin bands. On the basis of fractionation and subsequent characterization of the soluble antigen mixture by gel filtration, calcium phosphate chromatography, isoelectric precipitation, disc electrophoresis, and ultracentrifugation studies, two distinct classes of virus-induced antigens differing markedly in molecular weight were recognized. A high molecular weight class (200,000 and greater) contained at least three virus-induced antigens; a low molecular weight class (50,000 to 100,000 range) contained at least four immunoprecipitins. Further separation of the antigens within the two groups was accomplished. The two classes were distinguished also by their ability to stimulate synthesis of virus-neutralizing antibody. Antisera prepared against the high molecular weight class proved effective in neutralizing vaccinia virus. In contrast, the low molecular weight antigens showed little, if any, ability to induce formation of neutralizing antibody.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.     

Murine embryonal carcinoma cell-surface sialyl LeX is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl LeX was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (LeX or X hapten). Sialyl LeX was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl LeX and i antigens but not LeX, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.     

Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.