Chemical, Clinical, and Immunological Studies on the Products of Human Plasma Fractionation. Xl. Quantitative Separation and Determination of the Protein Components in Small Amounts of Normal Human Plasma 1

Lever, Walter F.; Gurd, Frank R. N.; Uroma, E; Brown, Roy; Barnes, Benjamin A.; Schmid, Karl; Schultz, Ethel L.

doi:10.1172/jci102423

Cited by 176 publications

(34 citation statements)

References 14 publications

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“…Streptokinase antibody assays were performed upon the isolated gamma globulin fractions of serum prepared by the technique of Lever and co-workers (12) and assayed by a modification of the Christensen technique (11). Clearance studies were performed on patients who required intravenous streptokinase for therapeutic reasons.…”

Section: Methodsmentioning

confidence: 99%

The Clearance of Heterologous Protein from the Circulation of Normal and Immunized Man12

Fletcher¹,

Alkjærsig²,

Sherry³

1958

J. Clin. Invest.

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The clearance rates of intravenously administered antigenic protein from the blood stream of normal and immunized animals have been studied by a number of observers and the results have been reviewed by Coons (1). Proteins labeled in various ways were used during these investigations, but the most relevant results appear to have been obtained when proteins were trace-labeled with radioisotopes (2), a technique resulting, under the best conditions, in little or no detectable change in the biological fate of the protein. In most instances the experimental conditions were such that the rate of protein removal was gradual and the period of observation extended over several days. However in one recent study (3) particular attention was paid to the changes occurring in the first hour or so after the injection of the antigenic material into the immune animal, a time during which antigen-antibody union in the circulation might be expected to exert a marked influence upon the clearance rate. Virtually all previous investigators have used the rabbit as a test animal and studies in man have not been reported.Streptokinase, a product of hemolytic streptococcal metabolism, when injected into man by the intravenous route and at appropriate dosage, will cause the activation of serum plasminogen and the occurrence of a fibrinolytic state (4). During the course of work aimed at inducing a state of controlled fibrinolysis in man for therapeutic reasons (5), the opportunity arose to determine the plasma clearance rates of streptokinase from the circulation of normal and immune man. Most streptococcal proteins are antigenic to man and strepto-

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Section: Methodsmentioning

confidence: 99%

The Clearance of Heterologous Protein from the Circulation of Normal and Immunized Man12

Fletcher¹,

Alkjærsig²,

Sherry³

1958

J. Clin. Invest.

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“…albumin plus alpha globulin, beta globulins and gamma globulins by the method of Lever, Gurd, Uroma, Brown, Barnes, Schmid and Schultz (1951 water (pH7). A precipitate formed which was then dissolved in 0-9 per cent NaCl or Hank's solution.…”

Section: Serum Fractionation Proceduresmentioning

confidence: 99%

The Agglutination of Tumour Cells In Vitro by Sera from Tumour Patients and Pregnant Women

Tal¹,

Dishon²,

Gross³

1964

Br J Cancer

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IN the course of study on the possibility of autoimmune mechanisms against tumour, the level of circulating antibodies to the autologous tumour cells was estimated by cell agglutination titres according to a technique previously developed in this laboratory (Pikovski, Tal, Schlesinger and Margoliash, 1957). It was soon found that cross agglutination occurred. It appeared that the sera from tumour patients contained a factor (AF) which could cause agglutination of, and be adsorbed by, tumour cells from a variety of sources. This paper represents an analysis of this phenomenon. MATERIALS AND METHODS Cell preparationCell suspensions were prepared from some human tumours, several normal human tissues and some mouse tumours by a previously described technique (Pikovski, Tal, Schlesinger and Margoliash, 1957). Suspensions of tissue cultures of HeLa cervical carcinoma, KB buccal carcinoma and Chang normal liver cells were also used. The preparation of the tissue culture suspensions is given in the following detail using HeLa cultures as the example.HeLa cultures were grown in Eagle's lact-yeast medium plus 15 per cent of normal human serum and horse serum, respectively, in milk bottles. After 7-8 days of growth, the medium was pipetted off, and the cell layer washed with 10 ml. of warm (370 C.) phosphate-buffered saline (PBS). The PBS was replaced by 10 ml. of 0-02 per cent Ethylenediamine Tetracetic acid (EDTA) in PBS devoid of Ca and Mg, and the culture kept at 370 C. for [15][16][17][18][19][20] minutes. This procedure loosened the cells from the wall of the culture bottle and the EDTA cell suspension was transferred to a test tube. Clumps of cells were dispersed by repeated pipetting with a fine Pasteur pipette, taking care to avoid foaming. The suspension was then centrifuged at 500 r.p.m. for 5 minutes. The supernate was carefully removed so as not to disturb the cell pellet. The cells were then resuspended in 8 ml. of warm RBS and centrifuged as above. The washed cells were counted and suspended in sufficient normal saline to give a concentration of 4 x 106 cells per ml. Agglutination procedureSera were diluted fourfold with 0-2 M phosphate buffer (pH 7.0). The pH of the system was critical. 2 x 105 Cells were added to 0 4 ml. of serum dilutions.

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“…This evidence will be presented in detail later. One of the important methods for differentiation of lipoproteins does not show differences between fractions in the proportions of free and esterified cholesterol (40).…”

Section: Resultsmentioning

confidence: 99%

The Study of Serum Proteins and Lipids With the Aid of the Quantity Ultracentrifuge. I. Procedure and Principal Features of the Centrifugate of Untreated Normal Serum as Determined by Quantitative Analysis of Samples From Ten Levels 12

Turner¹,

Snavely²,

Goldwater³

et al. 1951

J. Clin. Invest.

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The studies described in this paper and some which follow have as an objective a better description of serum lipids. The present paper represents the first detailed report of work begun early in 1948, some features of which have been published in abstract (1)(2)(3)(4)(5). Initially our purpose was to extend the observations of Recant, Chargaff and Hanger (6) on the influence of serum proteins upon the cephalin-cholesterol flocculation and the thymol turbidity tests. We included quantitative analyses of the centrifugate for lipids as well as for proteins. It soon became apparent that we were using a procedure which was useful in the study of the structure and function of lipid complexes.As shown in the review by Edsall (7) there has long been evidence that a large proportion of the lipids of the serum occur not as small molecules, but as lipid aggregates, or lipoproteins, of varying complexity. The use of the ultracentrifuge, both quantitative and analytical, has a well established place in the study of serum lipids. Important contributions have been made by McFarlane (8,9), Pedersen (10, 11), the Harvard group (12) ment of rate of flotation when suspended in a standard medium, a technic which we have not used. Principal emphasis in published studies by other workers has been on centrifugation of treated serum. In the present paper we deal exclusively with centrifugation of untreated whole serum. At present it does not seem profitable to attempt a detailed comparison between the results obtained by our procedure on the one hand and those obtained by the system of analysis introduced by Cohn and his associates (21), and the results of the flotation procedures of others. When our studies of diseased states and normal individuals under dietary stress have been presented, such comparisons will be in order. Some of the similarities of our contributions to those of others will be mentioned below in the discussion.The perfect procedure for the study of serum proteins and lipids in the initial step would give complete separation of the different lipid and protein complexes, each entity in pure state with structure unchanged by the procedure effecting separation. We doubt if the procedure described in this communication separates any single lipid or protein complex in pure form; nevertheless, the concentration of complexes at certain levels in the centrifugate is of such a degree as to give new information concerning the structure of the complexes at that level and their concentration in the serum under study. The lipid distribution curves are reproducible within satisfactory limits with normal sera, and are significantly different in certain diseased states, especially in acute virus hepatitis, as we have already reported briefly (3) and will report later in detail. We believe our procedure has three main advantages: The manipulation of serum which effects redistribution of pro-1071 TURNER, SNAVELY, GOLDWATER, RANDOLPH, SPRAGUE, AND UNGLAUB teins and lipids is gentle and involves minimal risk of damage to complex molecul...

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Chemical, Clinical, and Immunological Studies on the Products of Human Plasma Fractionation. Xl. Quantitative Separation and Determination of the Protein Components in Small Amounts of Normal Human Plasma 1

Cited by 176 publications

References 14 publications

The Clearance of Heterologous Protein from the Circulation of Normal and Immunized Man12

The Clearance of Heterologous Protein from the Circulation of Normal and Immunized Man12

The Agglutination of Tumour Cells In Vitro by Sera from Tumour Patients and Pregnant Women

The Study of Serum Proteins and Lipids With the Aid of the Quantity Ultracentrifuge. I. Procedure and Principal Features of the Centrifugate of Untreated Normal Serum as Determined by Quantitative Analysis of Samples From Ten Levels 12

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