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IMMUNOLOGICAL TECHNIQUES

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  • 1. IMMUNOLOGICAL TECHNIQUES BIOLOGY/CHEMISTRY 233 COURSE GREENSHEET FALL 2008 INSTRUCTOR: Tzvia Abramson, Ph.D. Office: DH 446 Contact: (408) 924-4872 (Office) (408) 924-4900 (Dept Office) Tzvia.Abramson@sjsu.edu Access on line: https://ewok.biology.sjsu.edu/DrAbramson User id: ewokbiostudents Password: 4biolecture Office hours: MW 11:30-1:00 by appointment COURSE DESCRIPTION: A laboratory/discussion course exploring the theoretical and practical aspects of contemporary immunological techniques. Students will be responsible for both oral and written material presented in laboratory discussions and in the course of conducting the laboratory exercises. This includes both the theoretical and practical aspects of applied immunology including understanding various methods and their applications, and interpretation of results. The goal of this course will be to provide students with some experience in methods used in immunology, particularly the use of specific antibody in biomolecular applications. The techniques should, however, be applicable in a number of diverse fields. PREREQUISITE: Biology 6 and Biology 107 (or equivalent) with a >B grade. Consent of instructor. TEXT: • Boothby: Immunological Techniques Laboratory Syllabus Biological Students Association (DH 346) 2008
  • 2. • Harlow & Lane: Antibodies (reference/recommended) Cold Springs Harbor Press 1988 • Abbas KA and Lichtman AH: Cellular and molecular immunology 6th edition 2007 Elsevier Saunders publishing 2005 • Janeway, et. al.: Immunobiology 7th edition (reference/recommended) Garland Science Publishing 2007 • Howard & Bethel - Basic methods in antibody production and characterization. CRCpress 2001 • Howard C Gary and Matthew R. Kaser . Making and using antibodies- a practical handbook by Gary C CRC 2007 SKILL LEVEL: It will be assumed that students entering this course will have taken upper division courses with laboratories in Immunology and Biochemistry. Material presented in these courses will not be reviewed. CLASS HOURS: This will be a lecture/laboratory course. The scheduled hours will be MW 2:30-7:00. Outside hours for nonhazardous activities can be arranged. Several activities in addition to scheduled exercises will be expected. EXAMINATIONS: There will be two examinations. Examinations will be given during the laboratory period (see schedule). The second examination may be given during the scheduled final examination time. Examinations will cover all appropriate course material and will emphasize comprehension and reasoning capabilities. Examinations are comprehensive. No late examinations will be accepted without prior permission from the instructor. Makeup exams will be oral. GRADES: The course grade will be based on the two examinations (30% each), presentations from the current literature (10% presentation/5% evaluations of presentations: total 15%), a laboratory notebook (15%), and a subjective evaluation by the instructor (10%) based on observed technical skills, understanding of lab procedures and safety instructions, and participation in student evaluations, activities and discussions. ii 2 2
  • 3. DEPARTMENT POLICY: University and department guidelines require a serious and compelling reason to drop a course. Grades, alone, do not constitute a reason to drop a course. LAB FEE: A lab fee is charged for this class at the time of registration. SJSU ACADEMIC SENATE POLICY: Academic integrity statement (from Office of Judicial Affairs): “Your own commitment to learning, as evidenced by your enrollment at San José State University, and the University’s Academic Integrity Policy requires you to be honest in all your academic course work. Faculty are required to report all infractions to the Office of Judicial Affairs. The policy on academic integrity can be found at (include url here). Campus policy in compliance with the Americans with Disabilities Act: “If you need course adaptations or accommodations because of a disability, or if you need special arrangements in case the building must be evacuated, please make an appointment with me as soon as possible, or see me during office hours. Presidential Directive 97-03 requires that students with disabilities register with DRC to establish a record of their disability.” LATE OR RETROACTIVE ADDS: It is the student's responsibility to determine that they are enrolled in courses. Official enrollment in a course should be determined on 'My SJSU' or by confirming with the course instructor BEFORE the last day to add. No late or retroactive add forms will be approved in the department office after the last day to add. iii 3 3
  • 4. TABLE OF CONTENTS Page Contents_____________________________________________ ii-iv Course Green Sheet ___________________________________ v-viii Laboratory Schedule __________________________________ ix-xi Laboratory Exercise # 1 ________________________________ 1 Introduction/Orientation Notebook format Laboratory Exercise # 2 ________________________________ 6 Preimune serum sample Immunize fusion mice (1°) Pristane prime mice Laboratory Exercise # 3 _________________________________ 8 Hybridoma assignment Harvest hybridoma cells Inject hybridoma for ascites production Laboratory Exercise # 4 ________________________________ 13 Thaw SP2/0 cells Feed/split SP2/0 Harvest hybridoma supernatant Laboratory Exercise # 5 ________________________________ 16 Prepare protein standards Laboratory Exercise # 6 ________________________________ 20 Feed/split SP2/0 Immunize fusion mice (2°) Laboratory Exercise # 7 ________________________________ 22 Freeze SP2/O Harvest ascites Harvest hybridoma supernatant iv 4 4
  • 5. Laboratory Exercise # 8/9 _______________________________ 24 Purification of immunoglobulin from mouse ascites and culture supernatant Laboratory Exercise # 10 _______________________________ 30 Analysis of IgG: Protein concentration Immunize fusion mice (3°) Laboratory Exercise # 11 ________________________________ 36 Isotype determination Laboratory Exercise # 12 ________________________________ 39 Thaw SP2/0 cells Feed/split SP2/0 Laboratory Exercise # 13 ________________________________ 41 Feed/split SP2/0 Laboratory Exercise # 14 ________________________________ 42 ELISA confirmation of immune response in fusion mice Feed/split SP2/0 Immunize fusion mice (4°) Laboratory Exercise # 15 ________________________________ 44 Fusion Laboratory Exercise #16 ________________________________ 48 Feed fusion Laboratory Exercise # 17 ________________________________ 49 Analysis of immunoglobulin: SDS-PAGE Laboratory Exercise # 18 ________________________________ 54 Analysis of Ig: SDS-PAGE..MW determination/densitometry Laboratory Exercise # 19 ________________________________ 57 Titration of purified antibody Laboratory Exercise # 20 ________________________________ 59 Specificity of purified antibody v 5 5
  • 6. Laboratory Exercise # 21 ________________________________ 61 Screen fusion by ELISA Laboratory Exercise # 22 ________________________________ 63 Clone hybrids from fusion Laboratory Exercise # 23________________________________ 66 Western blot --SDS PAGE/NCM transfer Laboratory Exercise # 24________________________________ 72 Immunoblot--Probe NCM Laboratory Exercise # 25________________________________ 74 Screen cloned hybridomas by ELISA BIOLOGY 233 FALL 2008 SCHEDULE EXC Date Production of hybridomas Purification and characterization Research of mAb paper prelab Aug 6? Prime mice with pristane (EXC-2B, p.6) 1/2 Aug 25 -Check in -Check in (M) -Obtain preimmune -Assign hybridoma (EXC--2D, p. serum (EXC-2c, p.6) 7) 3 Aug 27 -Immunize mice for -Harvest and count hybridoma (W) 0 fusion (1 ) (EXC-3B, p.8) cells (EXC-3C, p.10) -------------------- -Inject pristine primed mice with hybridoma cells for ascites production (EXC-3D, p.10) -------------------- -Split/feed hybridoma and harvest supernatant (EXC-4D, p. 15) - Aug29 -Split/feed hybridoma and (F) harvest supernatant (see EXC-4D, p.15) vi 6 6
  • 7. Sept 1 Labor day- no lab Labor day –no lab (M) 4/5 Sept 3 -Thaw SP2/0 (EXC-4B, p. -Split/feed hybridoma and (W) 13) harvest supernatant (see EXC-4D, p.15) -------------------- -Prepare/ evaluate protein standards (EXC-5BC, p.16) -Check/feed hybridoma and harvest supernatant - Sept 5 -Split/feed SP2/0 -Split/feed hybridoma and (F) (EXC-4C, p.14) harvest supernatant (see EXC-4D, p.15)ß 6 Sept 8 -Split/feed SP2/0 -Split/feed hybridoma and (M) (EXC-4C, p.14) harvest supernatant --------------------- (see EXC-4D, p.15) -Immunize mice for fusion (20) (EXC-6B, p.20) 7 Sept 10 -Freeze SP2/0 stock -Split/feed hybridoma and (W) (EXC-7B, p.22) harvest supernatant (see EXC-4D, p.15) -------------------- -Harvest ascites ? (EXC-7C, p.23) - Sept 12 -Split/feed hybridoma and (F) harvest supernatant (see EXC-4D, p.15) 8 Sept 15 -Purification of antibodies from (M) ascites and culture supt. (EXC-8B, p. 25) 9 Sept 17 -Purification of antibodies from Research (W) ascites and culture sup. paper (EXC-9B, p. 28) -Analysis of purified -Immunize mice for monoclonal antibody (mAb) and Research 10 Sept 22 fusion (30) (EXC-10C, p. protein concentration paper (M) 35) determination (EXC-10B,p.31) 11 Sept 24 -Determination of mAb isotype (W)* FACS (EXC-11B, p.37) Notebook vii 7 7
  • 8. 17 Sept 26 -Determination of monoclonal Research (M) purity by paper SDS-PAGE (run gel --> fix) (EXC-17, p.50) 18 Oct 1 -Determination of monoclonal Research (W) purity by paper SDS-PAGE (image analysis) (EXC-18, p.55) 12 Oct 6 -Thaw SP2/0 (EXC-12B, Research (M) p.40) paper x2 12 Oct 8 -Split/feed SP2/0 (W) (EXC-12C, p.41 13 Oct 10 -Check/feed SP2/0 Research (F) (EXC-13B, p.42) Paper x2 14 Oct 13 -Obtain immune serum (M) (EXC-2Cp.6) ----------------------------- -Determine mouse immune response by ELISA (EXC-14C, p.43) ---------------- -Check/feed SP2/0 (EXC-13B, p. 42) ---------------- -Immunize mice for fusion (40) IP (EXC-14D, p.44) -Immunize mice for fusion (40 -Immunize mice for fusion ( fusion mi -Immunize mice for fusion ( 15 Oct 15 FUSION (W) (EXC-15B, p.45) -Obtain final serum (Larry) - Oct 20 EXAM EXAM (M) viii 8 8
  • 9. 16 Oct 22 -Feed fusion (EXC-16B, Research (W) p.49) Paper 19 Oct 27 -Titrate purified antibodies by Research (M) ELISA (EXC-19, p.58) Paper x2 20 Oct 29 -Observe fusion plates Determination of purified mouse Research (W) antibody specificity using ELISA Paper x2 Notebook (EXC-20, p.60) 21 Nov 3 -Screen hybridoma by (M) ELISA (EXC-21, p.62) ----------------------- Prepare feeder cells (EXC-22B, p.64) 22 Nov 5 -Clone positive Research (W) hybridoma EXC- 22 C paper x2 Nov 10 Feed/screen/clone Research (M) paper x2 immunoblot analysis (SDS-PAGE) Nov 12 -Feed clones Research (W) Paper x2 immunoblot analysis (Probe blot - Feed clone -23 Nov 17 Feed/screen/clone Confirmation of antibody (M) specificity by (Western blot) (EXC-23C/D, p.69) 24 Nov 19 - Feed/screen/clone Western Blot (W) (EXC-24, p.73) 25 Nov 24 Feed/screen/clone Research (M) -------------------- paper x2 Freeze positive clones (EXC-7B, p.22) 26 Nov 26 Feed/screen/Transfer (W) clones/turn in - Dec 1 Feed/screen/Transfer (M) clones/turn in ix 9 9
  • 10. Dec 3* Feed/screen/Transfer (W) clones/turn in Notebook Dec 8 Final exam TBA (M) Dec 10 Check out Check out TBA • x 10 10