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| Preparation and Application of Immunoassay Signal Amplification Antibody Conjugates |
SHEN Yan,YAO Bin-yan,YANG Ruo-nan,KANG Wen-bin,LIN Hai-ying**( ) |
| Medicine Biotechnology and Engineering Research Institute, College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China |
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Abstract Objective: Chemical coupling agent and polylysine (PL) were used to conjugate goat anti-rabbit IgG and horseradish peroxidase (HRP) to increase the number of HRP connections on antibodies to amplify the sensitivity of immune detection, and the sensitivity was detected and compared through immunodetection applications, which is of great significance for subsequent immune diagnosis and biological research. Methods: Tylthioacetate (SATA) and Traut’s were optimized for the polymer HRP-PL, the feed ratio of Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) was optimized with goat anti-rabbit IgG and HRP-PL, and the reaction ratio of the two was optimized. The conjugated IgG-PL-HRP and commercial secondary antibody were detected by immunoblot, ELISA and immunohistochemistry, respectively, and the amplification of conjugated IgG-PL-HRP was calculated. Results: When the molar ratio of PL to HRP was 1∶5, the molar ratio of HRP-PL to Traut’s was 1∶15, the molar ratio of goat anti-rabbit IgG to Sulfo-SMCC was 1∶30, the molar ratio of goat anti-rabbit IgG to HRP-PL was 1∶10, the reaction efficiency was high. In the immune spot test, the minimum detection limits of commercial secondary antibody and conjugate IgG-PL-HRP were 2.5 μg and 312.5 ng, respectively, and the maximum dilution times were 50 and 100 times, respectively. In ELISA experiment, the maximum dilution times were 5 000 and 20 000, times respectively. In the immunohistochemical experiment, the detection specificity and intensity of conjugated IgG-PL-HRP were higher than those of commercial secondary antibody. Conclusion: The primary amplification conjugate IgG-PL-HRP was successfully synthesized and its immune detection signal amplification was about 3 ~ 7 times that of the commercial secondary antibody. It is of great significance to the following immunodiagnosis and biological research.
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Received: 18 July 2022
Published: 14 February 2023
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| [1] |
汤家宝, 李廷栋, 郭小怡, 等. 免疫检测信号放大技术研究进展. 中华微生物学和免疫学杂志, 2020, 40(2): 160-164.
|
|
|
| [1] |
Tang J B, Li T D, Guo X Y, et al. Research progress in signal amplification for immunoassays. Chinese Journal of Microbiology and Immunology, 2020, 40(2): 160-164.
|
|
|
| [2] |
Labib M, Hedström M, Amin M, et al. A capacitive biosensor for detection of staphylococcal enterotoxin B. Analytical and Bioanalytical Chemistry, 2009, 393(5): 1539-1544.
doi: 10.1007/s00216-008-2559-x
pmid: 19096830
|
|
|
| [3] |
Hayes D F, Bast R C, Desch C E, et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. JNCI: Journal of the National Cancer Institute, 1996, 88(20): 1456-1466.
doi: 10.1093/jnci/88.20.1456
|
|
|
| [4] |
Hashida S, Imagawa M, Inoue S, et al. More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge. Journal of Applied Biochemistry, 1984, 6(1-2): 56-63.
pmid: 6490581
|
|
|
| [5] |
Madersbacher S, Wolf H, Gerth R, et al. Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies. Journal of Immunological Methods, 1992, 152(1): 9-13.
pmid: 1379279
|
|
|
| [6] |
Presentini R, Terrana B. Influence of the antibody-peroxidase coupling methods on the conjugates stability and on the methodologies for the preservation of the activity in time. Journal of Immunoassay, 1995, 16(3): 309-324.
pmid: 7593652
|
|
|
| [7] |
Tsuruta J, Yamamoto T, Kozono K, et al. Application of a new method of antibody-enzyme conjugation with maleimide derivative for immunohistochemistry: hepatocellular production, interstitial tissue distribution, and renal cell reabsorption of plasma albumin in Guinea pig. The Journal of Histochemistry and Cytochemistry, 1985, 33(8): 767-777.
doi: 10.1177/33.8.3926865
|
|
|
| [8] |
Boorsma D M, Kalsbeek G L. A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method. The Journal of Histochemistry and Cytochemistry, 1975, 23(3): 200-207.
doi: 10.1177/23.3.47869
|
|
|
| [9] |
Boorsma D M, Streefkerk J G. Peroxidase-conjugate chromatography isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel. The Journal of Histochemistry and Cytochemistry, 1976, 24(3): 481-486.
doi: 10.1177/24.3.177698
|
|
|
| [10] |
Boorsma D M. Some aspects of immunoenzyme cytochemistry. Acta Histochemica Supplementband, 1988, 35: 41-51.
pmid: 3138719
|
|
|
| [11] |
Tuuminen T, Seppänen H, Pitkänen E M, et al. Improvement of immunoglobulin M capture immunoassay specificity: toxoplasma antibody detection method as a model. Journal of Clinical Microbiology, 1999, 37(1): 270-273.
pmid: 9854112
|
|
|
| [12] |
王朋飞, 徐胜楠, 魏杰, 等. 肽类树状大分子的功能化修饰及在肿瘤治疗和诊断中的应用综述. 材料导报, 2022, 36(11): 219-226.
|
|
|
| [12] |
Wang P F, Xu S N, Wei J, et al. Functionalization of peptide dendrimers and its application in tumor treatment and diagnosis: a review. Materials Reports, 2022, 36(11): 219-226.
|
|
|
| [13] |
Traut R R, Bollen A, Sun T T, et al. Methyl 4-mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the Escherichia coli 30S ribosome. Biochemistry, 1973, 12(17): 3266-3273.
pmid: 4581787
|
|
|
| [14] |
Liu M Y, Zhang X Q, Yang B, et al. Polylysine crosslinked AIE dye based fluorescent organic nanoparticles for biological imaging applications. Macromolecular Bioscience, 2014, 14(9): 1260-1267.
doi: 10.1002/mabi.201400140
pmid: 24854875
|
|
|
| [15] |
Pauli J, Grabolle M, Brehm R, et al. Suitable labels for molecular imaging - influence of dye structure and hydrophilicity on the spectroscopic properties of IgG conjugates. Bioconjugate Chemistry, 2011, 22(7): 1298-1308.
doi: 10.1021/bc1004763
|
|
|
| [16] |
Pauli J, Kai L C, Berkemeyer J, et al. New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging. Bioconjugate Chemistry, 2013, 24(7): 1174-1185.
doi: 10.1021/bc4000349
pmid: 23758616
|
|
|
| [17] |
Abedi M, Cohan R A, Mahboudi F, et al. Novel trastuzumab-DM1 conjugate: synthesis and bio-evaluation. Journal of Cellular Physiology, 2019, 234(10): 18206-18213.
doi: 10.1002/jcp.28453
pmid: 30854662
|
|
|
| [18] |
Berg E A, Fishman J B. Labeling antibodies using N-hydroxysuccinimide (NHS)-fluorescein. Cold Spring Harbor Protocols, 2019, 2019(3): t099283.
|
|
|
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