Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

Adamczyk, Maciej; Gebler, John C.; Mattingly, Phillip G.

doi:10.1021/bc960035h

Cited by 47 publications

(32 citation statements)

References 38 publications

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“…It has been observed that the higher antibody titer with moderate antibody affinities is obtained with hapten density of 15–30 molecules per carrier protein (for M3 conjugate). Similar observations have also been reported by other groups [14, 15] where it has been shown that the lower hapten density induces a slower immune response while higher substitution resulted in an IgM response exceeding that of IgG and produced antibodies of lower affinity.…”

Section: Resultssupporting

confidence: 91%

Characterization of Hapten–Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring

2013

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The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten–protein conjugates. In the present study, we report a new fluorescence-based method for the characterization of hapten–protein conjugates. The method is based on an effect promoted by hapten–protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten–protein conjugation density of two different class of pesticides (atrazine and 2,4-dichlorophenoxyacetic acid in this study) coupled to carrier protein. The study proved useful for monitoring the course of hapten–protein conjugation for the production of specific antibodies against small molecules. Well-characterized hapten–protein conjugates enabled obtaining highly sensitive anti-atrazine and anti-2,4-D antibodies with IC50 values equal to 12 and 70 ng mL−1 for atrazine and 2,4-D respectively. These antibodies were used for developing a fluorescence-based immunoassays format demonstrating a detection limit of atrazine and 2,4-D in standard water samples 2 and 7 ng mL−1, respectively. The developed immunoassay format could be used as convenient quantitative tools for sensitive and specific screening of pesticides in samples.

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Section: Resultssupporting

confidence: 91%

Characterization of Hapten–Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring

2013

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“…The higher ratio of hapten usually increases the strength and specificity of the immune response. However, there is a risk that a high degree of substitution could adversely affect the activity and specificity of antibodies produced [30]. At present, no official method for the determination of hapten-protein is available.…”

Section: Resultsmentioning

confidence: 99%

“…Generally, the evidence as to whether hapten has been successfully conjugated to the carrier protein is obtained after the putative conjugate is injected into laboratory animals and antibodies are detected, but this method is problematic, since it is time-consuming and wasteful [24]. Some methods have been tested in comparative studies [25,30], and UV is the technique commonly used for qualitation of the hapten-protein, including mycotoxins; however, this technique is not compatible in terms of the accuracy of FB 1 due to the lack of ultraviolet absorption functional group. MALDI-TOF-MS allows the determination of FB 1 -protein with good accuracy and precision and is appropriate for determining the other hapten-protein [31].…”

Section: Resultsmentioning

confidence: 99%

Characterization and Comparison of Fumonisin B1-Protein Conjugates by Six Methods

Wang

Zheng

et al. 2011

IJMS

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In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten (Fumonisin B1, FB1) was coupled to ovalbumin (OVA) and bovine serum albumin (BSA), respectively by a short cross-linker reagent (glutaraldehyde, GA). To develop a technique for detecting the conjugation, the hapten-protein conjugates (FB1-OVA and FB1-BSA) were characterized thoroughly by ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. The molecular weights of FB1-BSA and FB1-OVA were 74,355.301 Da and 48,009.212 Da, respectively determined by the method of MALDI-TOF-MS. The molecular coupling ratios were 11 and 5 in FB1-BSA and FB1-OVA, respectively. In this experiment, MALDI-TOF-MS was selected as the most efficient method to evaluate the cross-linking effect and calculate the molecular coupling ratio.

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“…The average number of A2E molecules per carrier protein in A2E-BSA and A2E-RSA conjugates was calculated by matrixassisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) (36,37). Fig.…”

Section: Resultsmentioning

confidence: 99%

Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells

Abeywickrama

Matsuda

Jockusch

et al. 2007

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

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The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photoexcited A2E.age-related macular degeneration (AMD) ͉ antibody ͉ immunocytochemistry

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Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

Cited by 47 publications

References 38 publications

Characterization of Hapten–Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring

Characterization of Hapten–Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring

Characterization and Comparison of Fumonisin B1-Protein Conjugates by Six Methods

Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells

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