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An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate

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An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-1981 An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate Allen H. Smith Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biochemistry Commons Recommended Citation Smith, Allen H., "An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate" (1981). All Graduate Theses and Dissertations. 5295. https://digitalcommons.usu.edu/etd/5295 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. AN ENZYME- LINKED ThltviUNOSORBE!'IT ASSAY (ELISA) FOR PANTOTHENATE by Allen H. Smith A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biochemistry UTAH STATE UNIVERSITY Logan, Utah 1981 ii ACKNOWLEDGEMENTS To Dr. R. G. Hansen, to Dr. B. W. Wyse, to Carl Wittwer, to Jack Brown, to Jan Pearson, to Nedra Christensen, to all those who have made this experience one of tremendous growth, I express my thanks. I express appreciation to the United States Department of Agriculture, under Grant #5901-0410-9-0288-0 with Utah State University, for financial support. Finally, I express thanks to my parents, who have come to realize that graduate school is also a part of life. Allen H. Smith "iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES . vi LIST OF FIGURES. vii · ABSTRACT .. ix INTRODUCTION 1 REVIEW OF LITERATURE 4 Pantothenate Assays . 4 ELISA: General . 5 ELISA: Detection . 6 ELISA: Quantitation .. 8 ELISA: Comprehensive Reviews 9 METI-IODS ... 10 Buffer Composition. 10 Nitrocellulose RIA. 11 Linear Range of the Gilford STASAR II Spectrophotometer at 405 Nanometers 12 Synthesis of Affinity Chromatography Support Material ......... 12 Purification of the Antibody .... 14 Stability of Antibody . 14 .Elution of Antibody from the Affinity ColUITU1. · · 15 Antibody Recovery in the Affinity Purification Process ....... 15 Synthesis of Multi-Pantothenate Human Serum Albumin-Pantothenate Conjugate. 16 Synthesis of MOno-Pantothenate Human Serum Albumin-Pantothenate Conjugate ..... 17 Alkaline Phosphatase-Antibody Conjugate Synthesis and Characteristics . 18 Indirect ELISA for Assay of Pantothenate: General Procedure . 19 Time Course of Color Development. 20 Determination of the Optimum Concentration of Human Serum Albumin-Pantothenate Conjugate Coating Culture Tubes ......... 21 Time Course of E-AB Competition for Free and Immobilized Pantothenate ......... 22 iv TABLE OF CONTENTS (continued) Page Comparison of ELISA and RIA Standard Curves . 23 Measurement of Food Extracts for Pantothenate via ELISA. 24 Measurement of Blood Extracts for Pantothenate via ELISA. 24 Optimum Conditions for ELISA Standard Curve 25 RESULTS. • . 27 Linear Range of the Gilford STASAR II · Spectrophotometer at 405 Nanometers ..... 27 Stability of Antibody in HOAC-DIOXANE . 27 Elution of Antibody from the Affinity Column 30 Antibody Recovery in the Affinity Purification Process. · · · · · 32 Synthesis of Multi-Pantothenate Human Serum Albumin-Pantothenate Conjugate . 32 Enzyme-Antibody Conjugate Characteristics . 34 Time Course of Color Development Enzyme­ Antibody Conjugate Immobilized on Polystyrene. 36 Determination of the Optimum Concentration of Human Serum Albumin Pantothenate Conjugate for Coating Polystyrene Culture Tubes .. 36 Time Course of E-AB Competition for Free and Immobilized Pantothenate . 41 Comparison of ELISA and RIA Standard Curves . 55 Measure of Food Extracts for Pantothenate via ELISA. 62 Measurement of Whole Blood Extracts for Pantothenate via ELISA . 64 Optimum ELISA Standard Curve. 65 DISCUSSION . 68 1>- fe~~od.S <Lid Procedures. 68 Major Findings .. 69 Discussion. 70 Conclusions . 75 Recommendations . 75 LITERATIJRE CITED . 7i 81 AP'Pf}ID ICES · · · · v TABLE OF CO'ITENTS (continued) Page Appendix A. Correction of Absorbance Data from the Non-linear Range of the STASAR II Spectrophotometer . Appendix B. Multi-PA HSA-PA Standard Curve Data .. Appendix C. Mono-PA HSA-PA Standard Curve Data. vi LIST OF TABLES Table -Page 1. Pantothenate-Binding Capacity of Protein Fractions from the Affinity Column ............ 30 2. Molar Ratio of P·antothenate to HSA in the Multi­ Pantothenate Human Serum Albumin Conjugate . 34 3. Optimt.nn Concentration of Multi-PA HSA-PA for Coating Culture Tubes ...... 41 4. ELISA Data for Comparison with RIA 56 5. RIA Data for Comparison with ELISA . 57 6. Assay of Food Extracts for Bantothenate via ELISA. 62 7. The Effect of Protein on ELISA ......... 64 8. Assay of Whole Blood Extracts for P~ntothenate via ELISA. 66 vii LIST OF FIGURES Figure Page 1. Linear range of the Gilford Stasar II Spectrophoto­ meter at 40S nanometers . 28 2. Stability of anti-pantothenate antibody in 0.1 N acetic acid + 10% 1,4-Dioxane solution ..... 29 3. Elution of antiserum from the affinity column with PBS and then with 0.1 N acetic acid+ 10%, 1,4- DioxaJle . 31 4. Polyacrylamide gel electrophoresis of protein fractions from the antibody purification regimen: (A) whole antiserum; (B) SO% saturated ammonium sulfate supernate; (C) SO% saturated ammonium sulfate precipitate; (D) PBS elution of the affinity column; (E) purified antibody. 33 S. Polyacrylamide gel electrophoresis of (A) purified anti-pantothenate antibody, (B) alkaline phosphatase, and (C) alkaline phosphatase-antibody conjugate 3S 6. Enzymatic activity of the alkaline phosphatase- antibody conjugate . 37 7. Enzymatic activity of alkaline-phosphatase-antibody conjugate attached to Multi-PA and Mono-PA HSA-PA adsorbed to polystyrene . 38 8. Determination of optimum concentration of Mono-PA HSA-PA for coating polystyrene culture tubes ... 40 9. Time course of color development; competition at room temperature using Multi-PA HSA-PA as the tube coating . 42 10. Time course of color development; competition at 4°C using Multi-PA HSA-PA as the tube coating . 43 11. Time course of color development; competition at room temperature using Mono-PA HSA-PA as the tube - coating . 44 12. T~e course of color development; competition at 4 C using Mono-PA HSA-PA as the tube coa'ting .... 4S 13. Stability of ELISA; competition at room temperature using ~fulti-PA HSA-PA as the tube coating ..... 46 viii LIST OF FIGURES (continued) Figure Page 14. Stability of ELISA; competition at 4°C using Multi- FA as the tube coating . 47 15. Stability of ELISA; competition at room temperature using Mono-PA HSA-PA as the tube coating . 48 16. Stability of ELISA; competition at 4°C using Mono- PA HSA-PA as the tube coating . 49 17. ELISA standard curve position as a function of compe­ tition time; competition at 40 C using Mbno-PA HSA-PA as the tube coating . 50 18. ELISA standard curve position as a function of compe­ tion time; competition at room temperature using Multi-PA HSA-PA as the tube coating. 51 19. ELISA standard curve position as a function of compe­ tition time; competition at 4°C using Multi-PA HSA-PA as the tube coating . 52 20. ELISA standard curve position as a function of competition time; competition at room temperature using Mono-PA HSA-PA as the tube coating . 53 21. Comparison of ELISA and RIA; standard curve plotted as the percent of pantothenate blank vs ng ng pantothenate . 58 22. Comparison of ELISA and RIA; standard curve plotted as the percent of pantothenate blank ·vs the log of ng pantothenate . 59 23. RIA standard curve (compare with figure 24) plotted as logit B/Bo vs the log of ng pantothenate. 60 24. ELISA standard curve (compare with figure 23) plotted as logit A/Ao vs the log of ng pantothenate 61 25. ELISA standard curves for assay of food extracts . 63 26. ELISA standard curve generated under opttmum conditions . 67 ix ABSTRACT An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate by Allen H. Smith, Master of Science Utah State University, 1981 Major Professor: Dr. R. Gaurth Hansen Department: Chemistry and Biochemistry An enzyme-linked immunosorbent assay (ELISA) for pantothenate has been developed. Antibodies induced in rabbits against bovine serum albumin-pantothenate conjugate were specifically purified by affinity chromatography. This process served to reduce the amount of endogenous pantothenate attached to the antibody, as well as to purify the antibody. The purified antibodies were covalently linked to alkaline phosphatase (Sigma type VII) with glutaraldehyde (0.05% aqueous solution). An immobilized pantothenate substrate was first obtained by attaching human serum albumin-pantothenate conjugate to the surface of polystyrene culture tubes by passive adsorption. The binding of the enzyme labelled antibody .CE: AB} to this substrate is propor­ tionately inhibited by free pantothenate as standards or as samples for analysis. The inhibition of E-AB immobilization was quantitated at 405 nm by the hydrolysis of p-nitrophenyl phosphate as indicated by the formation of p-nitrophenol. A standard curve was plotted on log logit paper, and was linear in the range of 2 through 1000 ng pantothenate. Initial experiments show that the ELISA will be use­ ful in assessing pantothenate in deproteinized blood samples and in food extracts. (85 pages) INrRODUCfiON Pantothenate is a vitamin found in nearly every biological system. It is an important component of Coenzyme A (Lipmann et al., 1950), a cofactor involved in energy metabolism,
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