Non-Immunological Precipitation of Serum by Sodium Dodecyl Sulfate in Agar Diffusion

Marmoset serum or serum of other species of animal may react with sodium dodecyl sulfate and forms nonspecific precipitin lines in agar diffusion. The protein detergent complexes are not readily dialyzable. Therefore precipitin lines derived from studies that use sodium dodecyl sulfate-treated antigens in agar diffusion must be interpreted with caution.     

Purification of pig renin

1. A new method of purification of renin is described. This method employs the following procedures: ethanol precipitation; saline extraction; precipitation of renin with 40% ammonium sulphate; precipitation of impurities with 3% ammonium sulphate at pH2.5; chromatography on DEAE-cellulose and CM-Sephadex; gel filtration on Sephadex G-100 (both normal and superfine grade); finally, starch-gel electrophoresis. 2. The final renin preparation had a specific activity 10(4) times that of the initial saline extract. 3. A single band of stained protein corresponding to the renin activity was present on starch-gel electrophoresis in the final step and a single precipitin line was obtained to this material with rabbit anti-(pig renin) serum. 4. Double diffusion in agar with rabbit anti-(pig renin) serum showed one major precipitin line, probably due to renin-anti-renin complex, and in addition two minor components.     

Nonspecific Precipitation of Serum Proteins by Sodium Lauryl Sulfate in Agar Diffusion and Immunoelectrophoresis

The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.     

STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

A detergent-independent procedure for the isolation of gap junctions from rat liver

In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.     

Affinity chromatography of two sets of isomeric antibodies having specificity for different oligosaccharide units of gum arabic

Two sets of antibodies directed against different carbohydrate units of gum arabic were isolated from the sera of rabbits immunized intramuscularly with gum arabic and Freund's complete adjuvant. The isolation was effected by affinity chromatography on two columns attached in series and containing an absorbent of AH-Sepharose 4B with ligands of partially hydrolyzed gun arabic in the first column and an adsorbent of AH-Sepharose 4B with ligands of native gum arabic in the second column. The two populations of anti-gum arabic antibodies were obtained and have been designated as Set 1 and Set 2 on the basis of their mobilities on agar diffusion. The antibodies of Set 1 consisted of 4 isomeric antibodies and those of Set 2 consisted of 11 isomeric antibodies. Native gum arabic samples were oxidized with periodate or reduced with sodium borohydride and carbodiimide under standard conditions and the modified samples were totally inactive in the precipitin test. On the basis of methylation data and immunological results it was concluded that terminal disaccharide moieties of the gum having the structure beta-D-glucosyluronic acid-(1----6)-D-galactose and alpha-L-arabinofuranosyl-(1----4)-D-glucuronic acid were the immunodeterminant groups for Set 1 and Set 2 antibodies, respectively.     

Mouse virulent strain of Staphylococcus epidermidis. Relation of antiphagocytic activity to the protection-inducing antigen.

Using 10(9) or 10(7) colony-forming units of a strain of Staphylococcus epidermidis (strain 1142) in saline or 5% mucin, respectively, 90 to 100% of mice died within 24 to 48 hr after intraperitoneal challenge infection. These organisms gradually multiplied in the peritoneal cavity when injected intraperitoneally into mice, while the mouse avirulent strain (strain 1124) rapidly decreased and no organisms were found there 20 hr after injection. This strain was capable of inducing resistance against challenge with homologous strains. The resistance appeared as early as the first week and disappeared the 4th week after the immunization. However, no resistance was induced with strain 1124 against challenge with strain 1142. Also, hyperimmune rabbit serum prepared with strain 1142 passively protected against challenge with homologous strain in mice. The protective antibody was absorbed out with homologous organisms but not with strain 1124. Subsequently, a surface substance was obtained from strains 1142 or 1124 by the method of Morse. The 1142 surface substance was capable of inducing a resistance against challenge with the homologous strain but not with the 1124 surface substance. Also, this substance absorbed the protective antibody in hyperimmune rabbit serum prepared with the homologous strain but not with the 1124 surface substance nor with the Smith surface antigen extracted from the Smith strain of Staphylococcus aureus. Conversely, the protective antibody in rabbit anti-Smith strain serum against challenge with the homologous strain was absorbed with the Smith surface antigen but not with the 1142 surface substance. In the agar diffusion test, the 1142 surface substance and the Smith surface antigen produced single precipitin lines only against homologous antisera. Biochemical analysis of the 1142 surface substance showed that the substance contained neither nucleic acids nor proteins but is composed of hexosamine, glycerol, phosphorus, alanine, glycine and phenylalanine.     

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.     

Antibodies specific to isoniazid and isonicotinic acid.

Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.     

Comparative Studies of Antigens from Mycoplasma mycoides and Escherichia coli

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.     

Micro diffusion precipitin tests for enteroviruses and influenza B virus

Middleton, G. K., Jr. (Bowman Gray School of Medicine, Winston-Salem, N.C.), H. G. Cramblett, H. L. Moffet, J. P. Black, and H. Shulenberger. Micro diffusion precipitin tests for enteroviruses and influenza B virus. J. Bacteriol. 87:1171-1176. 1964.-A simple micro precipitin gel diffusion test has been adapted to the study of viral antigens. As far as is known from a review of recent literature, this is the first use of the ECHO viruses in precipitin tests and the first attempt to demonstrate by the gel diffusion technique precipitins in a patient's serum after natural virus infection rather than artificial immunization. The principal value of this technique in virology is the rapid identification or qualitative analysis of viral antigen preparations by use of pooled or specific hyperimmune sera. Virus concentrations of 10(7)tcid(50) per 0.1 ml are required for reliable results, but only 0.015 ml of serum is necessary for each test. Virus-serum precipitin reactions were type-specific except for reciprocal precipitation of ECHO 1 and ECHO 8 by their hyperimmune sera. No viral antigens have been found common to two or more virus types among those tested. Precipitins for viral antigens occur frequently in serum of patients after a viral infection and are readily detected by micro precipitin gel diffusion tests. However, this precipitin test remains at present a tool for virus and antigen identification and offers an approach for research appraisal of host response to infections.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses from white-tailed deer blood samples dried on paper strips.

The feasibility of using dried blood samples for serologic testing of white-tailed deer (Odocoileus virginianus) for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was tested with matched samples of serum and eluted dried whole blood. Results from matched serum virus neutralization (SN) tests indicated that a 1-ml elution from a 1- x 2-cm section of filter paper strip containing dried blood approximated a 1:10 serum dilution. Neutralizing antibody titers detected from 34 matched titrations of serum and dried blood samples were equivalent in 25 (74%) titrations and were within a single dilution in the remaining nine (26%) titrations. Eluted blood samples from SN-positive deer, however, did not produce detectable precipitin lines on agar gel immunodiffusion tests for antibodies to either BTV or EHDV. In a trial using serum and dried blood samples from 108 hunter-killed deer from five locations in Georgia (USA), antibody prevalence and serotype distribution results were similar. Use of dried blood samples for serologic testing for antibodies to BTV and EHDV provides a reliable alternative to serum but should be considered only when serum collection is not feasible.     

Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.     

Production and characterization of DR xenoantisera: use for detection of serum DR antigens.

Immunoadsorbents consisting of rabbit anti-DR antibodies bound to Staphylococcus aureus were used to bind DR antigens from NP40 cellular extracts of cultured B lymphoid cells. When injected into rabbits, these DR antibody absorbents elicited the production of antibodies that were shown by both serologic and immunochemical techniques to react specifically with the DR antigen molecule. A rosette inhibition assay with B lymphoid cells and anti-DR xenoantisera was used to detect DR antigens in human serum. DR serum antigens could be found in an enriched preparation of serum lipoproteins obtained by centrifugation in KBr. In addition, sera from some patients with neoplastic diseases contained higher levels of DR serum antigens than those found in normal individuals.     

Immunochemical study of beta-glucuronidase inhibitor from porcine sublingual gland. Interaction of beta-glucuronidase inhibitor with alpha2-macroglobulin.

Antiserum to the inhibitor of beta=glucuronidase isolated from porcine sublingual gland was prepared in rabbits. Double immunodiffusion with the inhibitor produced a single precipitin line. However, neutralization of the inhibitor was produced by the antiserum and also by normal serum. Anti-beta-glucuronidase inhibitor isolated from human serum, by fractionation with (NH4)2 SO4 followed by DEAE-cellulose, Sephadex G-200 and Sepharose 4B chromatography, was identified as alpha2-macroglobulin by using ultracentrifuge analysis and immunoelectrophoresis. The mechanism of interaction of beta-glucuronidase inhibitor with alpha2-macroglobulin was also studied.     

An Agar Diffusion Method for the Determination Of Antibodies Against Staphylococcus Aureus Deoxyribonuclease

On the addition of small concentrations of deoxyribonuclease, produced by Staphylococcus aureus, to Toluidine Blue DNA agar, a medium is produced on which antibodies against S. aureus deoxyribonuclease may be detected. When samples of milk, or blood serum, containing antibodies against S. aureus are applied into wells in the agar, the deoxyribonuclease activity is inhibited by the antibodies diffusing into the agar. As a result of this inhibition, blue zones are produced around the wells in the otherwise bluish-red agar. The diameters of the zones correspond to the concentrations of antibodies, and the method may consequently be used for qualitative and quantitative examinations of antibodies against S. aureus deoxyribonuclease in milk and serum. The procedure and certain limitations of the method are described.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Serosurvey for Newcastle disease and avian influenza A virus antibodies in great cormorants from France

Inland great cormorants (Phalacrocorax carbo) culled in France were examined in the winter of 1997-98 and 1998-99 for antibodies to Newcastle disease (ND) and influenza A strains H5 and H7 by the hemagglutination inhibition test. Antibodies to influenza A group antigen were tested by agar gel precipitin test. Ten of 53 adult individuals were seropositive for ND virus. All sera were negative for influenza A antibodies. It is speculated that ND occurred in the sampled population.     

Chicken antichromatin antibodies: specificity to different chromatin fractions.

Specific complement-fixing and precipitin antibodies were induced in chicken to chromatin from WI-38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement-fixation reaction. These antibodies are species specific and they react at the complement-fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.     

Crossed immunoelectrophoretic analyses of antigens from Trypanosoma lewisi and Trypanosoma musculi

Trypanosoma lewisi and Trypanosoma musculi were collected from immunosuppressed infected hosts and extracted with phosphate buffered saline. Antisera were obtained from rats repeatedly infected with T. lewisi or mice repeatedly infected with T. musculi. Cellular antigens (CAg) present in the extracts of the parasites were analyzed by microimmunodiffusion (MID), crossed immunoelectrophoresis (CIE) and tandem crossed immunoelectrophoresis (TCIE). Trypanosome extracts were absorbed with the heterologous hyperimmune antisera to examine shared and unique antigens of the parasites.

Extracts of T. lewisi formed four precipitin lines when reacted with hyperimmune rat antiserum and three precipitin lines were detected by mouse anti-T. musculi serum in MID analyses. T. musculi extract formed two precipitin lines with mouse hyperimmune serum and two precipitin lines with rat anti-T. lewisi serum in the MID tests. When T. lewisi was reacted with the homologous hyperimmune rat antiserum in CIE, 14 precipitin peaks developed, while T. musculi extract formed eight peaks with homologous mouse hyperimmune serum. Seven precipitin peaks developed when T. lewisi extract was reacted with the mouse antiserum and T. musculi extract formed eight peaks during its electrophoretic migration into rat anti-T lewisi serum. TCIE clearly showed that five T. lewisi CAg could not be detected in the T. musculi extract by the rat antiserum, while mouse anti-T. musculi serum formed six precipitin peaks with the T. lewisi extract and seven peaks with the homologous extract. One of the CAg present in the T. musculi extract was not found in the T. lewisi extract. Absorptions of the extracts with heterologous antisera and subsequent CIE against the homologous antisera indicated three of the CAg of T. lewisi were not shared by T. musculi, while a single antigen of T. musculi was not detected in T. lewisi. Although concentrations of antibodies in each of the antisera and CAg in the parasite extracts were not equivalent, the data indicated that a minimum of eight CAg are shared by these rodent trypanosomes and at least three antigens appeared to be unique to T. lewisi and a single antigen to T. musculi.