Application of fluorescence polarization to the antigen-antibody reaction

Dandliker, Walter B.; Schapiro, Harriette C.; Meduski, J.W; Alonso, Richard; Feigen, George A.; Hamrick, J.R

doi:10.1016/0019-2791(64)90041-2

Cited by 115 publications

(33 citation statements)

References 21 publications

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“…The value obtained from five determinations is ~4 9 5 = 72000 f 7000 ern--' M-' at pH 2 8. This value is in reasonable agreement with the results of Dandliker et al [24] and Tengerdy and Chang [25] who found that the absorption coefficient of fluorescein covalently bound to a protein is lower by 12-18 % than that of free fluorescein, for which The main band located at 495 nm decreases upon lowering the pH following the ionization of a group with a pK of 6.20 -t 0.05 (Fig. 5).…”

Section: Molar Absorption Coefficientsupporting

confidence: 92%

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Garel

1976

Eur J Biochem

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A chemical derivative of bovine pancreatic ribonuclease A (RNase A) has been prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the a-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. It is shown that the pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2'-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied by mean of the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. It is proposed that using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.

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Section: Molar Absorption Coefficientsupporting

confidence: 92%

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Garel

1976

Eur J Biochem

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“…To further dissect antigenic requirements in our rabbit model we have utilized a hapten-carrier system. The fluorescein hapt en system [16,17] was selected for these investigations for its tracer properties and its immunogenic potency, attributed to its characteristic as a 'space-filling' anti genic moiety [18] which eliminates anomalous effects arising from heterogeneous linkages encountered with the use of smaller haptens [19]. The high affini ties (as much as 1014 liters/m) observed with antibod ies to fluorescein [20,21] are consistent with the size, aromaticity and dianionic nature of the fluorophore.…”

Section: Introductionmentioning

confidence: 99%

Carrier Requirement for Development of Acute Experimental Hypersensitivity Pneumonitis in the Rabbit

Butler¹,

Richerson

Swanson

et al. 1983

Int Arch Allergy Immunol

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Fluorescein isothiocyanate (FITC) conjugated to protein carriers was used to explore carrier dependence in an established rabbit model of acute hypersensitivity pneumonitis (HSP). Rabbits were immunized via toepads with either FITC-ovalbumin (OA) or FITC-human γ-globulin (HGG) in complete Freund’s adjuvant, and were aerosol challenged with homologous or heterologous conjugates 30 days later. Only those rabbits challenged with the homologous carrier developed acute HSP, despite the presence of comparable levels of anti-FITC antibodies in the sera of all groups. These findings indicate a strict carrier dependence in the pathogenesis of HSP in this model and provide further evidence that the mechanism of inflammation depends upon a cellular immune response.

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“…The concentration of oPRL-(1-53) was determined by using absorptivity E0-1% cm = 0.46, calculated by the method of Wetlaufer (11); the concentration of oPRL-(54-199) was determined by The peptide peak (-100 mg, 2% peptide) was lyophilized and redissolved in 10 ml of 0.01 M NH4HCO3 (pH 8.4) as a stock solution. Polarization experiments were carried out with [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Al of the stock solution plus 2 ml of the NH4HCO2 buffer. All labeling and storage procedures were carried out in darkness.…”

Section: Methodsmentioning

confidence: 99%

“…In order to investigate this apparent dichotomy, we have examined the interaction of the fibrinolysin fragments by using fluorescence polarization (5). Although this method has most commonly been applied to antigen-antibody binding (6,7), the principles involved can be generalized to study any reaction producing a sufficiently large molecular weight change in the fluorescent species (5,7,8). The usefulness of this technique has been greatly expanded in recent years by the development of appropriate instrumentation (9) and by the characterization of fluorescein isothiocyanate as a fluorescent label (10).…”

mentioning

confidence: 99%

Ovine prolactin: equilibrium characteristics of the recombinant molecule formed by noncovalent interaction of two fibrinolysin fragments by fluorescence polarization.

Jibson¹,

Li²,

Glaser³

1981

Proc. Natl. Acad. Sci. U.S.A.

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The equilibrium characteristics of the recombined molecule formed by noncovalent interaction of two fibrinolysin fragments of ovine prolactin (oPRL) have been studied with fluorescence polarization. Fluorescein isothiocyanate (isomer I) was used to label oPRL-(1-53), creating a fluorescent peptide indistinguishable from the unlabeled fragment inthe complementation reaction with oPRL-(54-199). The dissociation constant of the recombinant prolactin was 0.144 ,uM at 30°C, with a free energy of dissociation of 9.50 kcal/mol.

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Application of fluorescence polarization to the antigen-antibody reaction

Cited by 115 publications

References 21 publications

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Carrier Requirement for Development of Acute Experimental Hypersensitivity Pneumonitis in the Rabbit

Ovine prolactin: equilibrium characteristics of the recombinant molecule formed by noncovalent interaction of two fibrinolysin fragments by fluorescence polarization.

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