Species-Specificity of Human Anti-Beef, Pork Insulin Serum

Berson, Solomon A.; Yalow, Rosalyn S.

doi:10.1172/jci103980

Cited by 139 publications

(49 citation statements)

References 12 publications

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“…In contrast, although 125Tg B cells are anergic to BCR stimulation, they do not undergo developmental arrest (25); rather, they enter mature B cell subsets where they pose a risk by contributing to active immune responses. The presence of such B cells in the mature repertoire is consistent with the clinical observation that administration of autologous insulin routinely leads to the production of insulin Abs (26). Thus, a key issue for preventing autoimmunity is to understand how the threshold for tolerance is governed in self-reactive B cells that escape developmental arrest.…”

supporting

confidence: 65%

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

Acevedo-Suárez

Kilkenny

Reich³

et al. 2006

The Journal of Immunology

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B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.

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supporting

confidence: 65%

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

Acevedo-Suárez

Kilkenny

Reich³

et al. 2006

The Journal of Immunology

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“…Since both pH (7) and time of incubation (8) have a known effect on formation of insulin-antibody complexes, it was initially believed that differences in these or less obvious experimental conditions could have accounted for the different conclusions reached by the two investigations. In our experiments neither incubation at pH 7.4 nor incubation for 4 days at 40 C altered the results. Although Yalow and Berson noted that "the relative degree to which human insulin and beef insulin react at different insulin concentrations varies severalfold," these conclusions are difficult to reconcile with their own data, as shown in Figure 3.…”

Section: Binding Of Insulin To Antibodies Discussioncontrasting

confidence: 82%

Comparative Binding of Beef and Human Insulin to Insulin Antibodies Produced in Man and Guinea Pigs*

Grodsky¹,

Forsham²

1961

J. Clin. Invest.

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Two methods (1, 2) for the immunochemical assay of circulating human insulin have been described recently. Both are based on the displacement of insulin-I131 from antibody by the addition of unlabeled insulin but differ in the conditions employed for the combination of insulin to antib)o(ly and for the subsequent separation of the bound from free insulin. Beef insulin was used as the reference standard by Grodsky and Forsham (1) because a) human insulin of sufficient purity to allow determination of its specific insulin content by biological assay was not available, and b) it was shown that human and beef insulins bind to guinea pig antibeef insulin antibody in a proportional relationship. Yalow and Berson (2) criticized the use of beef insulin as a standard, stating that huiman and beef insulins react disproportionately with guinea pig antiserum.In other communications (3, 4), a 25-to 100-fold difference between the affinity of human insulin and beef insulin for antibodies produced in the human against antibeef-pork insulin was reported.The importance of these phenomena in the immunoassay of circulating insulin, as well as their effect on the metabolism of this hormone, has recently become apparent. On the basis of these considerations, we undertook to reinvestigate the quantitative relationship between beef and human insulin for binding to guinea pig and human antibody.

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“…Some authors proposed to draw asymptotic straight lines a t both extremities of the Sctachard plot [11,15] and the lines are moved parallel so that the sum of the ordinate intercepts equals the total ordinate intercept. It is also possible to calculate the intrinsic binding constants from the intercepts and limiting slopes the curve [16].…”

Section: Graphic Pabameter Pittingmentioning

confidence: 99%

Determination of Binding Parameters from Scatchard Plots

Weder

Schildknecht

Lutz

et al. 1974

European Journal of Biochemistry

146

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Procedures for graphic and numerical parameter fitting from Scatchard plots for the case of two different independent sets of equivalent binding sites are described. These methods are compared with other procedures and their advantages and disadvantages are discussed. Estimates of how much one parameter can be changed without changing the Scatchard curve significantly are made. This variability of the parameters is discussed. It is shown that low saturation points are of considerable importance in evaluating the binding parameters of the high affinity binding site. An example of the parameter fitting procedures is given and the utility and limitations of the procedures are discussed. It has been stated [6] that a logarithmic plot should be used to exclude other forms of data representation. The logarithmic scale allows one t o plot all theoretically obtainable data. The doublereciprocaI plot [7] and the Scott or half-reciprocal plot [8] have open upper limits on the abscissa as has the absorption isotherm. I n the Scatchard plot, all theoretically obtainable data can be plotted provided that the degree of binding is used on the abscissa and the nonspecific binding sites with large binding capacity are absent. An objection to the Sctachard plot was that linearisation of binding data, which is only possible in the rare case of only one single set of binding sites, should result in a loss of information [4]. Nevertheless, we studied the representation of binding data with the Scatchard plot, mainly because of its wide-spead use, but being all the time aware of its drawbacks in certain cases.A similar study has been undertaken by Feldman 191, where a least-squares parameter fitting has been used for the numerical method. Least-squares parameter fitting was also used by Nagy [lo]. A graphic parameter fitting procedure has been described by Berson and Yalow [ll], and Rosenthal [12], while others [13,14] used a semiautomated parameter fitting on a small computer. The aim of the present investigation was to study the determination of the parameters of binding systems with different means of expenditure. The graphical and numerical curve fitting have been studied using a programmable calculator and a computer. The numerical parameter fitting of the computer program does not use least-squares regression, therefore some difficulties could be overcome. To test the applicability of the fitting procedure a practical example will be given as an illustration. BASIC RELATIONSHIPSThe outlined procedures are based on the following assumptions : all ligands are identical, the activity (chemical, biological) will not be altered except by binding, two independent sets of binding sites are present, all sites of each set are equivalent (identical), there is no cooperativity, each set of binding sites obeys the mass law action.At equilibrium for each system containing one single set of binding sites, the Scatchard plot [3] gives a straight line following the equation Eur. J. Biochem. 42 (1974)

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Species-Specificity of Human Anti-Beef, Pork Insulin Serum

Cited by 139 publications

References 12 publications

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

Comparative Binding of Beef and Human Insulin to Insulin Antibodies Produced in Man and Guinea Pigs*

Determination of Binding Parameters from Scatchard Plots

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