Preparation of conjugates of 19s and 7s globulins of antitularemic sera for the determination of specific fluorescence ofPasteurella tularensis

Pasteurella tularensis was detected by means of conjugates of 19S and 7S globulins from antitularemic serum with fluorescein isothiocyanate (FITC). The serum was obtained by short-term immunization of rabbits. The capacity of both fractions to produce the immunofluorescence reaction with pure culturesof Pasteurella tularensis and the agglutinin titre of these fractions are compared here. It was found that the 19S globulin fraction which contained most agglutinating antibodies showed only weak immunofluorescence. The 7S fraction in which the agglutinin content was very low as compared with the 19S globulin fraction gave more intense fluorescence than the 19S fraction. On the basis of this finding the opinion is advanced that the agglutinating antibodies need not be the actual carrier of fluorescence due to fluorescent antibodies (FA) in the immunofluorescence reaction ofPasteurella tularensis. The different degrees of labelling with FITC of the individual globulin fractions as observed during simultaneous conjugation are also discussed. The 19S globulin fraction is regularly conjugated to a higher degree than the 7S component. The view is presented that the avirulent strains lack a certain part of the antigen structure as compared with virulent or vaccine strains (with residual virulence), the part being responsible for the positive reaction of immunofluorescence with preparations of fluorescent antibodies.     

Criteria for Preparing, Evaluating, and Standardizing Iodinated Globulins for Radioimmunoassay Procedures

Procedures were examined for labeling immune globulins with radioactive iodine using chloramine-T as the oxidizing agent. The chloramine-T method was critically evaluated to establish the optimal conditions for preparing iodinated globulins with high specific radioactivities without impairing their immunospecificities for use in in vitro radioimmunoassays. The results showed that the use of 100 mug of chloramine-T per ml, 500 to 1,000 muCi of Na (125)I per mg of protein, and a 10-min oxidation reaction time produced globulins of both high specific radioactivities and immunospecificities. Criteria were established for evaluating and determining optimal concentrations of iodine-labeled globulin for use in radioimmunoassays. The results of this investigation indicated that the amount of labeled indicator globulin used in radioimmunoassays should be based upon protein concentration rather than radioactivity.     

Common Antigen(s) in Cotton to Fusarium oxysporum f. sp. vasinfectum

Antigen-antibody reactions in agar gel, as demonstrated by the double diffusion technique, between cotton seed globulins and the antisera specific to each of the tested Fusarium oxysporum f. sp. vasinfectum isolates as well as the antiserum of F. moniliforme revealed that all the tested antisera of F. oxysporum f. sp. vasinfectum reacted with seed globulins except the Menoufi cultivar globulins. No precipitin lines were detected in the reaction between the antigenof the cotton cultivar Acala SJ2 versus the antiserum of P10 isolate. The 5 cultivars behaved differently with each fungal antiserum to the extent that they could be distinguished accordingly. When the seed globulins of the susceptible cultivars (Giza 74, and Bahtim 110) reacted with antiserum of the tested F. oxysporum f. sp. vasinfectum isolates, more precipitin lines were formed than the resistant cultivars. On the other hand, no obvious reaction was detected in case of F. moniliforme antiserum.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.     

Identification of Actinomyces israelii and Actinomyces naeslundii by Fluorescent-Antibody and Agar-Gel Diffusion Techniques

This study was an attempt to develop a fluorescent-antibody (FA) test to differentiate Actinomyces israelii and A. naeslundii as an aid in their laboratory identification. Two strains of A. israelii (X522 and A601) and two strains of A. naeslundii (X454 and X600), which had received intensive study by several investigators, were used for the immunization of rabbits. Working titers, based on tests with antigens prepared from the homologous strains and from well-established heterologous strains, were determined for each labeled antibody preparation. These conjugates and their normal serum control conjugates were used separately to stain 85 cultures of Actinomyes species and 23 strains of other species that might be confused with them. Acetone-precipitated soluble antigens from these same strains were tested with different antisera in the agar-gel diffusion test. Results showed that A. israelii (X522 and A601) and A. naeslundii (X454 and X600) labeled antiglobulins, when used at their working titers, stained most strains of their homologous species. Agar-gel diffusion results showed general agreement with those of the FA tests. The two tests appear to be equal in sensitivity, but the FA test is more specific, since several cross-reactions were noted with the agar-gel diffusion test whereas no cross-reactions were obtained with the FA reagents. Agar-gel and FA studies suggest that at least two serotypes of A. israelii may be associated with human disease. Although the majority of strains tested in this study appear to belong to a common serotype, "serotype 1," two strains of an apparent second serotype, "serotype 2," were encountered. FA staining of tissue impression smears from experimentally infected mice was successful when a counterstain, Evans Blue dye, was used.     

Neisseria gonorrhoeae Identification in Direct Smears by a Fluorescent Antibody-Counterstain Method

Direct smears from female patients have been considered unreliable for the detection of Neisseria gonorrhoeae by fluorescent-antibody (FA) methods because of inadequate background contrast of the fluorescent-stained smears and a scarcity of organisms on the smear. Evans blue dye employed as a counterstain eliminated the nonspecific background staining and increased the reliability of the direct FA procedure. Direct smears demonstrating positive fluorescence were obtained from 86% of a group of culturally positive named female contacts. The FA-counterstain technique is as sensitive as the presently recommended cultural procedures.     

Solid Phase Radioimmunoassay for Identification of Herpesvirus hominis Types 1 and 2 from Clinical Materials

A solid phase radioimmunoassay (RIA) was developed for typing Herpesvirus hominis (HVH) strains isolated from clinical materials, and it also proved to be applicable to the direct detection and typing of HVH antigen in human and animal brain tissue. The procedure utilized virus-infected human fetal diploid cells or brain tissue smears in the bottom of 1-dram glass vials, antigen was detected through the use of intermediate HVH antisera produced in rabbits or hamsters and cross-absorbed with the HVH heterotype, and (125)I-labeled anti-species (rabbit or hamster) globulins produced in goats were used for detection of immune complexes. The cross-absorbed HVH antisera could be used at high dilutions in the RIA test, and they reacted with marked type-specificity in the RIA system. Specificity of the test was also improved by determining and using optimal concentrations of intermediate sera and of (125)I-labeled anti-species globulins. Results of typing HVH isolates by the RIA procedure agreed in all instances with those obtained by direct fluorescent antibody staining with cross-absorbed conjugates. The RIA procedure was effective and more sensitive than direct fluorescent antibody for demonstrating and typing HVH antigen directly in smears of infected human brain tissue.     

Passive Cutaneous Anaphylaxis with Antigens from Coxiella burneti

Passive cutaneous anaphylaxis (PCA) was produced in guinea pigs sensitized with guinea pig Coxiella burneti phase I–II antiserum and challenged with dimethylsulfoxide- or trichloroacetic acid-soluble extracts from phase I cells. The PCA reaction could not be induced by whole or mechanically disrupted phase I or phase II C. burneti cells or by extracted cells or extracts of phase II cells. The antibody responsible for PCA was in the 7Sγ₁ (fast γ) globulin. Sensitization of the skin by 7Sγ₁ antibody could be blocked nonspecifically by 7Sγ₁ globulin from normal serum or from phase II antiserum. The 7Sγ₂ (slow γ) globulin antibody inhibited the reaction specifically. Some antiserum pools containing high agglutinin and complement-fixing titers to phase I C. burneti cells failed to initiate the PCA reaction, perhaps due to an imbalanced ratio of γ₁ to γ₂ specific globulins or to an imbalance in the ratio of specific to nonspecific γ₁ globulins. Agglutinins to phase I cells were found in both γ₁ and γ₂ antibody globulins. Complement-fixing antibodies were found in the γ₂ globulin fraction.     

Preparation and characterization of the plasma membrane of pig lymphocytes

Lymphocyte plasma membrane was isolated from minced pig mesenteric lymph node by differential centrifugation and by centrifuging through a sucrose density gradient. The yield of membrane was approx. 0.1% (dry wt. relative to wet wt. of lymph node). The purified material had a sucrose density of 1.14g/cm(3) and consisted mainly of smooth vesicles. The membrane fraction contained, apart from protein and lipid, 59mug of carbohydrate, 11mug of sialic acid and 28mug of RNA/mg of protein; no DNA was detected. The cholesterol/phospholipid molar ratio was 1.01. Specific activities (mumol of product/h per mg of protein) of 5'-nucleotidase, succinate dehydrogenase, acid phosphatase and glucose 6-phosphatase were 10.1, 0, 0.51 and 0.30 respectively. The membrane vesicles were aggregated by an antiserum against pig lymphocytes and adsorbed the agglutinins to whole lymphocytes present in the antiserum; the membrane fraction was 28 times as effective as whole cells (on a dry wt. basis) in removing the lympho-agglutinins. Antisera against the membrane fraction agglutinated whole lymphocytes. It is concluded that the preparation represents the plasma membrane of small lymphocytes. The plasma membrane of pig thymocytes was isolated by using the same procedure. Its properties were similar to those of the lymphocyte plasma membrane.     

Identification of Homologous Globulins from Embryos of Wheat, Barley, Rye and Oats

Total globulins from embryos and endosperms of barley, wheat,rye, and oats were separated by SDS-PAGE under reducing andnon-reducing conditions. The preparations from embryos of allfour cereals contained major groups of bands with M_r's of 50-60000,which were not affected by reduction. These have been characterizedpreviously from oats and shown to correspond to subunits ofthe 7S storage globulin. Immunochemical relationships betweenthese bands (and others with M_r's between 40000 and 70000) weredemonstrated by immunodiffusion and ‘Western Blotting’using antiserum raised against the major subunits of the oat7S globulins. The 7S globulins were also prepared from hand-dissectedembryos of the four cereals using sucrose density ultracentrifugation.Their amino acid compositions were broadly similar, but differedfrom those of the 7S vicilins of legumes. It is concluded thatstructurally-related 7S globulins are present in the embryosof the four species of cereals. Key words: Homologous globulins, embryo, wheat, barley, rye, oats     

Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation

Bovine and human thyroxine-binding globulin were purified from serum by a three-step purification procedure which comprised affinity chromatography consecutively on thyroxine- and Concanavalin A--Sepharose and finally preparative polyacrylamide gel electrophoresis. The molecular weights of the two proteins were similar (54 000) as well as their carbohydrate contents while some differences in amino acid composition were found. Rabbit antiserum against bovine thyroxine-binding globulin reacted with human thyroxine-binding globulin with no sign of spur formation.     

THE EFFECT OF LABELING INTENSITY,ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY,ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES1

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.     

Identification of Actinomyces, Arachnia, Bacterionema, Rothia, and Propionibacterium Species by Defined Immunofluorescence

Fractionated fluorescein-isothiocyanate (FITC)-conjugated immunoglobulin G (dye-to-protein ratio <10), produced against whole cells of Actinomyces spp., Arachnia, Bacterionena, Rothia, and Propionibacterium spp., give species-specific conjugates with controlled nonspecific staining reactions when appropriately diluted on the basis of their antibody content (10 mg/ml). Using this standardization in immunofluorescence, serotype-specific conjugates are also available after dilution for all serotypes of these organisms except for Actinomyces viscosus type 2, and Propionibacterium acnes type 1. Adequately adsorbed conjugates could be used to differentiate these serotypes from A. viscosus type 1 and P. acnes type 2, respectively. A serological classification in defined immunofluorescence corresponded to species and serotype designation proposed on the basis of other serological analysis and biochemical characteristics. This includes a separation in immunofluorescence of two serotypes of Propionibacterium acnes. The detection of certain actinomycetes of the family Actinomycetaceae and Propionibacterium species by the defined immunofluorescence in direct smears prepared from clinical specimens agreed to 88% with parallel culturing when including a prereduced (PRAS) medium technique for isolation. Qualitative studies revealed that single cells of these organisms could be specifically identified by immunofluorescence when admixed with morphologically similar bacteria and a large number of other contaminants.     

Antibody to immunoselected L-cell antigens mimics stimulating activity of antibody to whole L cells

A heterogeneous IgG antibody raised in rabbits in response to injections of whole L cells was used to identify and select relevant antigens in a nonionic detergent extract of L cells prelabeled with [35S]methionine by means of immunoprecipitation and immunoaffinity chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the immunoprecipitate and immunoeluate contained far fewer protein bands than the whole cell extract but selectively retained a 42,000-MW protein species. In response to injections of the immunoprecipitate, rabbits produced a new antiserum which reacted predominantly with the 42,000-MW protein when reacted with L-cell proteins separated by sodium dodecyl sulfate-gel electrophoresis and transferred to nitrocellulose paper by the Western blot technique. The new antiserum (raised to the immunoprecipitate) and the original antiserum (raised to whole cells) were equipotent in stimulating calcium transport, phospholipid metabolism, and DNA synthesis in L cells. Binding of the IgG fractions of the two antisera displayed identical high affinity binding to L-cell surface antigens, with the same average association constant of 1.5 X 10(6) M-1. These studies have shown that an antiserum raised to whole L cells has a much narrower reactive spectrum with L-cell membrane antigens than might be imagined and has identified a 42,000-MW membrane protein as an important immunogen which itself elicits a potent immune response resulting in an antibody capable of mimicking the cell stimulatory properties of the original antiserum.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.     

Simplified Procedure for Preparation of Specific Antibodies to Gamma Globulins

Antibodies were prepared to rhesus monkey, rabbit, dog, and hamster gamma globulins which had been purified by agar-block electrophoresis. Immunoelectrophoretic analysis demonstrated that these antisera were specific for gamma globulin components in whole serum. The use of these antisera as secondary serological reagents was examined.     

Globulins enhance in vitro iron but not zinc dialysability: a study on six legume species.

The study was addressed to evaluate the in vitro iron and zinc dialysability from the globulin fraction of six legumes. Five legume species including white bean, mottled bean (Taylor bean), chickpea, lentil, lupin, and a modified mottled bean variety, selected by back-crossing to obtain seeds with globulins composed by G1 fraction only, were used. Globulins (G1 + G2) were extracted from the seeds and analysed for their in vitro iron and zinc dialysability. The highest globulin concentration was detected in lentil (89%). The percentage of globulins in the modified variety of Taylor bean (G1 only) was higher than that of the commercial variety (G1 + G2). The highest concentration of iron was found in Taylor bean globulins. The modified variety of Taylor bean contained 2.6-fold higher iron concentration than the whole seed, and the commercial variety had 1.8-fold higher iron only. The highest zinc concentration was found in lentil globulins. Also iron dialysability from globulins was markedly higher than that of the respective whole seed. The highest value of iron dialysability was found in lentil (10.8%). Zinc dialysability was generally high (above 20%), but no significant differences between whole seed and globulins were detected. The results showed that globulins enhanced iron but not zinc dialysability. Lupin and the modified variety of Taylor bean showed a different behaviour in terms of mineral dialysability compared to the other legumes. The amino acid composition of the digestion products of whole seeds and globulins failed to evidence any direct influence on iron and zinc availability.     

Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.     

Endocrine and reproductive responses of beef cattle to a synthetic gonadotropin-releasing hormone agonist (fertirelin acetate)

A synthetic gonadotropin-releasing hormone (GnRH) agonist (fertirelin acetate, FA) was administered to beef cattle within 12 h after onset of estrus (Day = 0) to study effects on subsequent endocrine responses and fertility. In Study 1, 16 crossbred beef heifers were injected with either 100 mug FA (n = 8) or saline (n = 8) at 6 or 12 h (n = 7; n = 9) after onset of estrus. Concentrations of luteinizing hormone (LH) over time were affected (P<0.01) by the interaction of treatment and interval from onset of estrus to treatment. Heifers treated with FA at 6 h after onset of estrus exhibited the greatest increase in LH after treatment. There was no effect of treatment, interval from onset of estrus to treatment or treatment by interval interaction on duration of the estrous cycle, on concentrations of progesterone from Days 1 through 14 posttreatment, or on concentrations of progesterone prior to subsequent estrus (Day -10 through 0, posttreatment estrus). In summary, FA administered to beef cattle within 12 h after onset of estrus effectively increased peripheral plasma concentration of LH, but this increase had no effect on subsequent luteal function as measured by duration of the estrous cycle or concentrations of plasma progesterone. In Study 2, 86 parous beef cows were bred artificially to one of two bulls following natural or prostaglandin F(2)alpha induced estrus. Cows received either no treatment or 50 or 100 mug FA at the time of AI. There was no effect of treatment, breed, parity, technician, service sire or interactions on conception rate (mean = 76.7%). Although not significant, the numerical pattern of conception rate among experimental groups (control = 71.4%, 50 mug FA = 76.7%, 100 mug FA = 82.1%) supports further investigation of this GnRH agonist with larger numbers of cattle.