Cent Saturated Ammonium Sulfate Research Articles

Defective monocyte leukotaxis in sarcoidosis: possible relationship to a plasma factor.

Monocyte leukotactic function was studied in 25 untreated patients with histologically confirmed sarcoidosis. Monocyte leukotactic responses were significantly depressed (P less than 0.001), most strikingly in patients with stage I disease; however, the severity of the leukotactic defect did not correlate with duration, activity, or extrathoracic dissemination of disease. Preincubation of normal monocytes in sarcoid plasma, but not normal plasma, resulted in partial inhibition of leukotactic responsiveness. The leukotactic inhibition was not reversed by washing the cells after preincubation or by subsequent exposure to normal plasma. The inhibitory activity, which was found in all sarcoid plasma samples, was nondialyzable, was heat stable, and could be localized to the 25 to 35 per cent saturated ammonium sulfate fraction of plasma. Lesser amounts of similar inhibitory activity were detected in comparable fractions of normal plasma. A highly significant correlation between monocyte leukotactic responses and plasma leukotactic inhibitory activity was found in patients with sarcoidosis, suggesting an important in vivo modulatory nole for this substance.

Crystallization and preliminary x-ray investigation of soybean beta-amylase.

Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52.

The primary interaction of antibody to components of aspergilli: II. Antibodies in sera from normal persons and from patients with aspergillosis

The capacity of antibodies in 102 human sera to bind components of aspergilli was studied. Sera were from patients with pulmonary aspergillosis or a variety of other clinical disorders and from healthy individuals. The antigens employed were anionic mucopolysaccharide components derived from A. fumigatus and A. nidulans and were not precipitated by antibodies in either human or rabbit sera. The antigens were soluble in 10 per cent trichloroacetic add and 50 per cent saturated ammonium sulfate, and antigen binding was measured by radioimmunodiffusion and the ammonium sulfate test. Sera from patients with aspergillosis, particularly the mycetomal form, bound significantly greater amounts of the antigens than did the other sera. Binding was associated with IgM and IgG, was to the F(ab)′2 portion of immunoglobulin, and was immunologically specific. Inhibition studies demonstrated the presence of shared antigens between aspergilli and a variety of other fungi, mycobacteria, non-acid-fast bacteria, and commercial house dust concentrates. The minimal but significant binding observed with sera from normal individuals is probably the result of antigenic stimulation by aspergilli as well as other fungi and organisms in the environment.

Tumor-associated antigen in patients with carcinoma of the colon

In 1965 Gold and Freedman [1,2] described an antigen isolated from saline extracts of colonic adenocarcinema. They called this antigen carcinoembryonic antigen (CEA) because of its distribution in neoplastic tissue of gastrointestinal origin and in the embryonic gastrointestinal tract. It was characterized by Krupey, Gold, and Freedman [3] as a protein polysaccharide complex soluble in 1.0 M perchloric acid (PCA) and 50 per cent saturated ammonium sulfate, and they have also reported its amino acid and carbohydrate composition. A radioimmunoassay procedure has been developed by Thomson et al [5] to detect and measure circulating antigen in the serum of patients. They demonstrated circulating CEA in a high percentage of patients with primary gastrointestinal neoplasms but they could not detect this antigen in patients with other types of primary neoplasms [5]. An antigen thought to be identical to CEA has been reported by Von Kleist and Burtin [4]. Serologic studies by Gold demonstrated antibodies to CEA in pregnant women and in a high percentage of patients with digestive system neoplasms both with and without metastases. For the past three years we have been attempting, in our laboratory, to further define the nature and significance of CEA as defined by Gold and Freedman [1,2]. Using both their reagents and methods and

Quantification of IgG by the ammonium sulfate technique—I. Interference of the primary interaction between 125I Fc and rabbit anti-human Fc

A sensitive, reproducible method to quantitate human IgG, utilizing 125I Fc, derived from either the papain sensitive (ps) or papain resistance (pr) conformational forms of IgG, as the test antigen in the ammonium sulfate technique is described. Since 125I Fc fragments so obtained are innately soluble in 45 per cent saturated ammonium sulfate, free antigen may be separated from bound antigen-antibody complexes. The test system takes advantage of the capacity of IgG containing materials to block the interaction between human 125I Fc and a standard rabbit anti-human Fc.

Measurement of serum DNA-binding activity in systemic lupus erythematosus.

Antibodies to DNA were demonstrated in the serums of patients with systemic lupus erythematosus (SLE) by ammonium sulfate precipitation. DNA is soluble in 50 per cent saturated ammonium sulfate, whereas immunoglobulins and immunoglobulin-bound DNA are insoluble. When ammonium sulfate is added to a mixture of radioactive DNA and serum, the precipitate contains radioactivity if DNA is bound to immunoglobulins. Abnormal binding was found in serum of 75 per cent of unselected patients with SLE, 25 per cent with Sjögren's syndrome, 5 per cent with related disease and 2 per cent of normal subjects. All of 52 selected SLE serums with positive, as well as 21 of 32 SLE serums with negative, complement-fixation tests for anti-DNA antibodies had abnormal binding. Binding activity was associated with immunoglobulin G of serum. High binding values were seen chiefly in patients with active SLE renal disease; marked reductions accompanied clinical improvement. The test overcomes problems inherent in other methods to detect anti-DNA antibodies, is easy to perform and is useful in the diagnosis and assessment of the course of SLE.

Antibody studies in rabbit encephalomyelitis induced by a water-soluble protein fraction of rabbit cord.

A water-soluble protein fraction of nervous tissue was prepared by extraction of rabbit spinal cord with sodium citrate at pH 4.3. Characterization by nitrogen content and paper electrophoresis showed it to be a mixture of basic proteins. This extract demonstrated encephalitogenic activity when injected into rabbits. The most suitable technique for the measurement of serum antibody to the rabbit cord antigen proved to be the precipitation of antigen-antibody complexes by 40 per cent saturated ammonium sulfate. Antibody could not be demonstrated by the techniques of complement fixation, quantitative precipitation, and Ouchterlony plates. The early appearance of circulating antibody occurred almost exclusively in rabbits that subsequently developed EAE. Specificity of the antibodies for nervous tissue was demonstrated by appropriate blocking and adsorption experiments.

Studies on the binding between streptococcal M protein and antibody.

A method for detecting type-specific antibody to type 12 beta-hemolytic streptococci is described. The method is based on the utilization of a partially purified I(131) labeled streptococcal acid extract and the solubility of this extract in 40 per cent saturated ammonium sulfate as compared to the insolubility of antibody and antigen-antibody complexes at this salt concentration. The salient features of this technique are: (a) When absorbed sera were used, no non-type-specific reaction with antigen occurred. (b) The sensitivity of the system allowed detection of type-specific antibody in hyperimmune antisera that had been diluted 1200-fold. (c) The method does not rely on secondary manifestations of antigen-antibody interaction for the detection of antibody. (d) As little as 0.03 microg of M(12) protein was detected when soluble unlabeled M protein was used to inhibit the reaction between I(131) M(12) protein and anti-M(12) antibody. (e) The kinetics of the reaction between M protein and anti-M antibody can be studied, thereby making information available as to the quality as well as the quantity of antibody produced in this antigen-antibody system.

Primary interaction between I 131 labeled ragweed pollen and antibodies in the sera of humans and rabbits

1. 1. Ragweed pollen extract was fractionated with 50 per cent saturated ammonium sulfate (SAS) and the supernatant labeled with I 131. 2. 2. When 0.02 γ N of this heterogenous mixture (I∗RW) was incubated with normal human or rabbit sera, approximately 10 per cent was nonspecifically precipitated with the globulins after the addition of 40 per cent SAS. 3. 3. After incubation with sera from humans or rabbits immunized with ragweed pollen extract as much as 40 to 60 per cent of the I∗RW was precipitated in 40 per cent SAS. 4. 4. Lesser degrees of increased precipitation of I∗RW were demonstrated with as little as 0.5 ml. of a 1:100 ragweed-injected human antiserum or 0.5 ml. of a 1:2000 ragweed-immunized rabbit antiserum. 5. 5. That the increased I∗RW precipitated by immune sera after the addition of 40 per cent SAS was due to binding by specific antibody rather than nonspecific co-precipitation was evidenced by: (a) rabbit antisera of high titer for bovine albumin, bovine globulin, human globulin, and Type 12 streptococcal M-protein precipitated no more I∗RW than did normal rabbit serum; (b) excess unlabeled ragweed pollen antigens completely inhibited the binding of I∗RW by human or rabbit anti-ragweed sera; and (c) apparently unrelated antigens did not inhibit the reaction. 6. 6. Determinations of the precipitation of I∗RW on different occasions by a pair of selected control sera demonstrated the reproducibility of the method and the stability of the labeled extract.

The separation of substances in timothy pollen extract producing allergic skin reactions from those producing hemagglutination reactions

The hemagglutination of rabbit red blood cells coupled to timothy pollen extract by diazo bonds was explored as in vitro assay for timothy allergen during fractionation and purification procedures. We found that rabbit red blood cells coupled to the fraction of timothy pollen precipitated by 20 per cent saturated ammonium sulfate were agglutinated by sera of patients allergic to timothy pollen, but that this fraction gave very weak skin test reactions. The fraction soluble in 49 per cent but precipitated by 55 per cent saturated ammonium sulfate gave low hemagglutination titers, but strongly positive skin tests. Passive transfer tests indicated that the hemagglutinating factor is distinct from reagin and blocking antibody. The separation of the fraction in timothy pollen producing hemagglutination from the fraction producing the skin reaction indicated that reagin and the antigen in timothy pollen responsible for the skin reaction are not involved in the hemagglutination reaction.

Studies on the pathogenesis of fever. VII. Preliminary chemical characterization of leucocytic pyrogen.

Study of the chemical properties of the pyrogenic component of rabbit polymorphonuclear leucocytes reveals it to contain an essential, non-dialyzable protein which: (a) is precipitated by perchloric acid, (b) is removed by extraction with phenol, (c) is soluble in 50 per cent methanol and 33 per cent saturated ammonium sulfate, and (d) is destroyed by the proteolytic action of both trypsin and pepsin. By combined chemical and chromatographic techniques the leucocytic pyrogen has been purified approximately 50-fold. The partially purified material contains less than 1 per cent carbohydrate, is resistant to periodate oxidation, is unaffected by extraction with butanol and contains at least two immunologically active components when tested by the Ouchterlony gel-diffusion technique. Its chemical properties distinguish it from other known pyrogenic substances which have been implicated in the pathogenesis of fever.

Aspermatogenesis, anaphylaxis, and cutaneous sensitization induced in the guinea pig by homologous testicular extract.

Guinea pig testicles were extracted with acetic acid; the extract was purified by removing material in consecutive precipitations with 30 per cent saturated ammonium-sulfate, trichloracetic acid, and chloroform. The solution so purified, when administered with complete adjuvants, was highly active in inducing impairment of spermatogenesis in guinea pigs. The activity resisted autoclaving at 15 pounds' pressure for 20 minutes, proteolytic enzymes, and formamide. Anaphylactic shock and cutaneous reaction to the purified homologous extract occurred in guinea pigs sensitized by the extract combined with adjuvants. For the production of aspermatogenesis it was essential to incorporate killed mycobacteria into the water-in-oil emulsion containing the antigen; but anaphylactic sensitization did not require the presence of mycobacteria.

Plasma Thromboplastin Component (PTC) A Hitherto Unrecognized Blood Coagulation Factor Case Report of PTC Deficiency

Abstract 1. A new hemorrhagic disease characterized by a prolonged whole blood coagulation time caused by the delayed formation of thrombin is described. 2. The patient suffering from this disease was found to have normal concentrations of all previously described coagulation factors. 3. Because the coagulation defect could be completely corrected by the addition of tissue thromboplastin or platelet-poor hemophilic plasma and because tests for prothrombin and prothrombin conversion accelerators gave normal results and prothrombin utilization was impaired, it was concluded that the missing factor was concerned with the production of thromboplastin in the plasma. Accordingly it was named the plasma thromboplastin component (PTC). 4. The clinical and laboratory resemblance of PTC deficiency and hemophilia is striking. However, the two diseases can be distinguished by the fact that normal serum, the 45 to 50 per cent saturated ammonium sulfate fraction of normal plasma, the citrate eluate from barium sulfate, which has been mixed with normal plasma or serum, and hemophilic plasma correct PTC deficiency but are ineffective in hemophilia. On the other hand, while the 33 per cent saturated ammonium sulfate fraction of normal plasma, barium sulfate adsorbed normal plasma, Cohn's Fraction I and PTC-deficient plasma correct the hemophilic defect, they are ineffective in PTC deficiency. 5. A method for the partial purification and concentration of the PTC factor is described.

Immunological studies on insect metamorphosis. I. Qualitative and quantitative description of the blood antigens of the Cecropia silkworm.

1. The cell-free blood of the Cecropia silkworm produces a maximum of nine bands of antigen-antibody precipitate when reacted in antiserum-agar tests with antisera prepared by injecting Cecropia extracts into rabbits. The blood antigens producing these bands of precipitate have the properties of proteins in that they are non-dialyzable, labile at 75 degrees C., and salted out by 75 per cent saturated ammonium sulfate. One antigen was identified as a carotenoid protein. 2. Six bands of precipitate were selected for further study. Absorption tests revealed that the blood, at all stages of metamorphosis, is capable of precipitating the antibodies which produce five of these bands. This result indicates that five of the six antigens are present in the blood throughout metamorphosis. The sixth antigen is undetectable in blood from fourth instar larvae, appears in the blood late in the fifth instar, persists during the pupal stage, and disappears again during adult development. 3. When blood samples from various stages of metamorphosis were tested in antiserum-agar tubes, the rates of advance of the six bands of precipitate underwent systematic change in close correlation with the morphological stage of the silkworm's metamorphosis. These changes are interpreted in terms of concentration changes of the corresponding blood antigens. The validity of this interpretation was tested in several ways, with the conclusion that the interpretation was generally acceptable for the system under consideration. 4. All six antigens appear to increase in concentration during the last larval instar and to decrease in concentration during the period of adult development. However, each antigen has its own characteristic pattern of concentration change which differs from those of the other five. In order to explain this diversity, we conclude that the physiological mechanisms which regulate the synthesis and utilization of the blood antigens control each antigen on an individual basis.

On the occurrence of renin in the blood of hypertensive patients

Renin free of hypertensinase has been extracted from lyophilized plasma by aid of a 40 per cent saturated ammonium sulfate solution. Application of this method to twenty-one patients with hypertension gave seventeen negative, one moderately positive, and three slightly positive results. The moderately positive blood was withdrawn from a patient with acute cardiac decompensation and probably cannot be ascribed to the earlier hypertension. The three slightly positive cases indicate quantities of renin that are much too small to elicit even a minimal pressor effect on intravenous injection.