Determination of precipitating titers and diffusion coefficients by double diffusion in gels

Zivkovic, T Dragana; Pokrić, Biserka; Pučar, Z.

doi:10.1021/ac50003a034

Cited by 8 publications

(5 citation statements)

References 23 publications

(9 reference statements)

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“…Since the critical precipitating concentrations are equimolar, and the "equivalency rule" demands equivalency at the onset of precipitation under conditions of diffusion [3,4,30,31 ], we may conclude that CaHPO4 is the first-formed precipitate at least in the phase of nucleation. This result does not imply that all phosphate in the buffer (pH 7.4) should be in HPO4"-ionic form; it indicates only that one calcium ion interacts with one phosphate ion during phase of nucleation.…”

Section: Precipitation Discsmentioning

confidence: 99%

Precipitation of calcium phosphates under conditions of double diffusion in collagen and gels of gelatin and agar

Pokrić

Pučar

1979

Calcif Tissue Int

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One-dimensional double diffusion was applied to determine critical concentrations at which the precipitation of calcium phosphates occurs in reconstituted connective tissue collagen and agar gels at 37 degrees C and in gelatin gels at 25 degrees C. Experiments were performed in the presence of unbuffered 0.15 mol dm-3 NaCl, or 0.15 mol dm-3 NaCl-veronal adjusted to pH 7.4. It was found that critical concentrations of precipitation of both precipitating components, CaCl2 and phosphate buffer (pH 7.4), were equimolar and independent of the ratios of initial concentrations of the components. Critical concentrations of precipitation were not affected by the concentrations and kinds of gels used. The first-formed precipitates showed amorphous structure by X-ray diffraction analyses. Infrared (IR) spectra of the precipitates indicated CaHPO4 . H2O to be their predominant species. The molar Ca/P ratio obtained by chemical analyses was 1.08. This precipitate transformed in time into octacalcium phosphate. In all experiments, two very thin membranes of precipitate were formed in the gel column at the onset of precipitation simultaneously on both sides of the actual disc of precipitate. IR spectra and chemical analyses showed that both membranes were identical to the actual precipitation discs.

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Section: Precipitation Discsmentioning

confidence: 99%

Precipitation of calcium phosphates under conditions of double diffusion in collagen and gels of gelatin and agar

Pokrić

Pučar

1979

Calcif Tissue Int

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“…In the case that the concentrations of the antigen and antibody solutions are unknown, the equivalence conditions can be determined by a rectangular two-dimensional double-diffusion technique called the “two-cross” immunodiffusion. A detailed description of the two-cross experimental setup which enables an adequate solution of Fick's second law of diffusion applied to the immunoprecipitation in gels is described elsewhere. , Briefly, a “cross” consists of four troughs cut at a right angle in a gel plate. The half-width of the trough is denoted by h .…”

Section: Theoretical Considerationsmentioning

confidence: 99%

“…According to the equivalence rule, the precipitation under conditions of a double diffusion starts at the equivalent molar concentrations of both precipitating components, ,− as indicated by eq 2: …”

Section: Theoretical Considerationsmentioning

confidence: 99%

“…A detailed description of the two-cross experimental setup which enables an adequate solution of Fick's second law of diffusion applied to the immunoprecipitation in gels is described elsewhere. 6,7 Briefly, a "cross" consists of four troughs cut at a right angle in a gel plate. The half-width of the trough is denoted by h. The troughs of each cross are filled in alternate order with antigen solution (component A) and immune serum (com-ponent B).…”

Section: Theoretical Considerationsmentioning

confidence: 99%

“…The immunodiffusion experiments were carried out at 20 and 40 °C. For all technical details concerning the two-cross immunodiffusion experimental procedure and the evaluation of the results, refer to Pokric ´and Puc ˇar 6 or Z Ä ivkovic ´et al 7 for somewhat less-detailed descriptions.…”

Section: Immunogen and Antigenmentioning

confidence: 99%

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Precipitation at Equivalence and Equilibrium: A Method for the Determination of Equilibrium Constants of Reaction between Multideterminant Antigen and Specific Polyclonal Antibodies

Pokrić

2000

J. Chem. Inf. Comput. Sci.

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A theoretical approach for the determination of the equilibrium constant, Ka, of the reaction between a multideterminant antigen (Ag) and specific polyclonal antibodies (Ab) forming the insoluble Ab/Ag immune complex, is derived. The constant can be expressed as a function of the two accessible experimental parameters, the precipitating concentration of the antigen and the Ab/Ag molar ratio. For this purpose Ab/Ag immune complex must be prepared at equivalence, and equilibrium between precipitated and soluble species must be reached. The proposed method is experimentally tested on the system human serum albumin (HSA) and polyclonal rabbit antibodies. The Ab/Ag precipitates are prepared by the direct mixing of biological fluids in which immunoreacting components naturally occur. Previous separation, purification, or labeling of immunoreacting components are not required. The conditions for the precipitation of Ab/Ag complexes at equivalence, the stoichiometric composition or the average number of Ab molecules bound to one Ag molecule, and the solubility of the immunoprecipitating components are determined by a rectangular two-dimensional double immunodiffusion. Since the solubility determined under the conditions of a double immunodiffusion is a result of the interaction of the global diffusion of the precipitating components and particle growth kinetics, it mostly refers to the dynamic conditions. To find the solubility under equilibrium conditions, it is sufficient to determine the minimal factor by which the solutions of both immunoprecipitating components should be diluted so that no precipitate is formed upon their mixing at equivalence. The dilution factor is determined by a measurement of the laser light scattering of the immunoprecipitating systems prepared with serially diluted Ag and Ab solutions.

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Dependence of diffusion coefficients and immunoprecipitating titers on pH: Albumin and IgG in human serum and their rabbit antibodies

Pokrić

Pučar

1980

Analytical Biochemistry

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Determination of precipitating titers and diffusion coefficients by double diffusion in gels

Cited by 8 publications

References 23 publications

Precipitation of calcium phosphates under conditions of double diffusion in collagen and gels of gelatin and agar

Precipitation of calcium phosphates under conditions of double diffusion in collagen and gels of gelatin and agar

Precipitation at Equivalence and Equilibrium: A Method for the Determination of Equilibrium Constants of Reaction between Multideterminant Antigen and Specific Polyclonal Antibodies

Dependence of diffusion coefficients and immunoprecipitating titers on pH: Albumin and IgG in human serum and their rabbit antibodies

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