Clinical Chemistry and Other Laboratory Tests on Mouse Plasma or Serum

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Besides hematological analyses, many other parameters, including clinical chemistry and endocrinological values, can be determined from mouse blood samples. For most of these tests, plasma or serum samples are used. Data obtained by these investigations provide indications of genotype effects on metabolism and organ functions. Here we describe in detail the considerations that have to be taken into account to get adequate samples for plasma or serum analyses and the recommended sample processing for different investigations. Furthermore, we describe established methods used in the German Mouse Clinic (GMC) to determine clinical chemical parameters; for more in?depth analysis of specific classes of biomarkers, we provide instructions for ELISAs (sandwich and competitive) as well as LC?MS/MS, focusing on markers associated with bone or steroid metabolism in the mouse as working examples. Curr. Protoc. Mouse Biol. 3:69?100 © 2013 by John Wiley & Sons, Inc.

Keywords: mouse; clinical chemistry; steroids; osteocalcin; PINP; ELISA; mass spectrometry

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Introduction
Basic Protocol 1: Serum Preparation from Mouse Blood
Basic Protocol 2: Plasma Preparation from Mouse Blood
Basic Protocol 3: Analysis of Clinical‐Chemical Parameters in Mouse Plasma Using the AU480 System
Basic Protocol 4: Measurement of Mouse Osteocalcin by a Commercially Available Sandwich ELISA (Direct Detection)
Basic Protocol 5: Measurement of Mouse/Rat PINP by a Commercial Available Competitive ELISA
Basic Protocol 6: Quantification of Steroids in Mouse Plasma Using Online SPE Coupled to LC‐MS/MS
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Serum Preparation from Mouse Blood

Materials

Mice
Plain 1.5‐ml cone‐shaped sample tubes for sample collection (e.g., Eppendorf; alternatively, tubes containing clot activator, with or without separating gel, can be used)
Rack for sample tubes
Tabletop microcentrifuge (e.g., Biofuge fresco, Heraeus)
1.5‐ml cone‐shaped sample tubes for separated serum (e.g., Eppendorf)

Additional reagents and equipment for blood collection from mice (Rathkolb et al., )

Basic Protocol 2: Plasma Preparation from Mouse Blood

Materials

Mice
0.5‐ to 1.0‐ml sample tubes for plasma preparation, containing an anticoagulant (e.g., Eppendorf; whether Li‐heparin‐coated, K‐EDTA‐coated, or citrated tubes are used depends on the analyses planned)
Rack for sample tubes
Tabletop microcentrifuge, preferentially coolable (e.g., Biofuge fresco)
1.5‐ml cone‐shaped sample tubes for separated plasma

Additional reagents and equipment for blood collection from mice (Rathkolb et al., )

Basic Protocol 3: Analysis of Clinical‐Chemical Parameters in Mouse Plasma Using the AU480 System

Materials

Mice
Deionized water supply providing sufficient amounts of deionized water to run the clinical chemistry analyzer [e.g., Medica water purification system, Elga LabWater (http://www.elgalabwater.com/), VWS‐Deutschland GmbH (http://www.veoliawaterst.de/)]
Reagents and solutions needed for operation of the AU480 autoanalyzer (Beckman Coulter):
- Wash solution (cat. no. OSR0001)
- Cleaning solution (cat. no. ODR3021)
- ISE buffer (cat. no. OE66320)
- ISE selectivity check (cat. no.OE66313)
- ISE Reference (cat. no. OE66318)
- ISE Mid Standard (cat. no. OE66319)
- ISE internal reference solution (cat. no. OE66314)
- Calibrators (see Tables 13.0.4300 to 13.0.4300 )
- Quality control samples (see Tables 13.0.4300 to 13.0.4300 )
- Test kits for the parameters to be analyzed (see Tables 13.0.4300 to 13.0.4300 )

Plain, Li‐heparin‐, or EDTA‐coated sample tubes (e.g., from Kabe Labortechnik, http://www.kabe‐labortechnik.de/: Li 500 A or Li 1000 A Standrand, EDTA 500 A or EDTA 1000 A Standrand) for blood collection
1.5‐ml cone‐shaped sample tubes for separated plasma
AU480 autoanalyzer (Beckman‐Coulter)
Software for data evaluation (see below)

Additional reagents and equipment for blood collection from mice (Rathkolb et al., ) and preparation of plasma ( protocol 2 ) or serum ( protocol 1 )

Basic Protocol 4: Measurement of Mouse Osteocalcin by a Commercially Available Sandwich ELISA (Direct Detection)

Materials

Osteocalcin sandwich ELISA kit (Biomedical Technologies, cat. no. BT‐470) including:
- Sample buffer, one 60‐ml bottle
- Wash buffer, phosphate‐saline concentrate, 5×, one 100 ml bottle: transfer contents to a graduated cylinder, bring volume up to 500 ml with deionized water, and mix
- Mouse osteocalcin standard, one vial, 200 ng, lyophilized: reconstitute standard with exactly 2.0 ml of sample buffer (yields a 100 ng/ml solution); use for making working standards; store reconstituted standard at −20°C
- Mouse osteocalcin antiserum (biotinylated osteocalcin, antibody specific for the C‐terminus), one 12‐ml vial
- Streptavidin–horseradish peroxidase, one 11‐ml vial
- Peroxidase substrate TMB (3,3¹ ,5,5¹ ‐tetramethyl benzidine), one 6‐ml vial
- Hydrogen peroxide Solution, one 6‐ml vial
- Stop solution (contains hydrochloric and phosphoric acids), one 12‐ml vial
- One 96‐well plate (8‐well removable strips) coated with a polyclonal osteocalcin antibody directed against the N‐terminus
- Adhesive plate sealer
Unknown samples: serum ( protocol 1 ) or plasma ( protocol 2 )

ELISA plate washer (optional)
ELISA plate reader that can measure absorbance at 450 nm

CAUTION: Some components of this kit contain isothiazolones (5 ppm) as preservative. Stop solution contains hydrochloric and phosphoric acids. Keep all materials away from the skin and eyes.

Basic Protocol 5: Measurement of Mouse/Rat PINP by a Commercial Available Competitive ELISA

Materials

Lyophilized rat/mouse PINP calibrators 0 to 5 and controls 1 and 2 (from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/): reconstitute calibrators and controls with 0.5 ml of distilled or deionized water, replace stopper, and let stand 5 to 10 min at room temperature; invert several times to ensure complete reconstitution (the exact value of each calibrator is printed on the bottle label; store reconstituted calibrators and controls at −20°C)
Unknown samples: serum ( protocol 1 ) or plasma ( protocol 2 )
Sample Diluent (20 ml per bottle; from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)
PINP biotin: lyophilized phosphate‐buffered saline containing PINP labeled with biotin, and BSA; 1 ml per bottle (from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)—reconstitute with 8 ml of Sample Diluent, replace stopper, and let stand for 5 to 10 min at room temperature; invert several times to ensure complete reconstitution
20× Wash Solution, Phosphate‐Saline Concentrate (50 ml per bottle; from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/): transfer contents to a graduated cylinder, bring volume up to 1000 ml with distilled or deionized water, and mix
Enzyme Conjugate: phosphate‐buffered saline containing avidin linked to horseradish peroxidase, protein, enzyme stabilizers and preservative (18 ml per bottle; from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)
TMB Substrate: aqueous formulation of TMB (3,3¹ ,5,5¹ ‐tetramethyl benzidine) and hydrogen peroxide (24 ml per bottle; from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)
Stop Solution: 0.5 M hydrochloric acid (14 ml per bottle; from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)

96‐well plate (8 well removable strips) coated with polyclonal rabbit anti‐PINP antibody (from Competitive ELISA kit; IDS Ltd, http://www.idsplc.com/)
Precision multichannel pipettes to deliver 45 µl, 50 µl, and 150 µl
Adhesive plate sealers
Microplate shaker
Automatic microplate washer (optional)
ELISA Plate Reader that can measure absorbance at 450 nm (reference 650 nm)

Basic Protocol 6: Quantification of Steroids in Mouse Plasma Using Online SPE Coupled to LC‐MS/MS

Materials

Mice
Norit A (activated charcoal, practical grade; Serva)
[¹³ C₂ ]testosterone (Cambridge Isotope Laboratories, https://www.cdnisotopes.com/)
d8‐corticosterone (C/D/N Isotopes, https://www.cdnisotopes.com/)
Methanol (HPLC grade, Merck)
ZnSO₄ (p.a., Merck)
Testosterone (Sigma)
Corticosterone (Sigma)
Androstenedione (Steraloids, http://steraloids.com/)
Acetonitrile (HPLC grade, Roth, http://www.carlroth.com/)
Formic acid (mass spectrometry, Fluka Analytical)
Double‐distilled water (or HPLC grade)

Centrifuges (Beckmann Avanti J 20, Hettich UNIVERSAL 32 R)
50‐ml conical tubes (e.g., BD Falcon)
Vortexer (Vortex‐Genie 2 or VWR Scientific DVX‐2500 Multi‐Tube Vortexer)
Labinco LD‐79 rotator (http://www.labinco‐bv.com/)
Sterile syringe filter (0.22 µm, Millipore)
Microbalance (e.g., Sartorius ME36S)
10‐ 50‐, and 100‐ml narrow‐necked volumetric flasks
1.5‐ml polypropylene safe‐lock reaction vials (e.g., Fisher Scientific)
Heidolph Reax 2 overhead shaker (http://www.heidolph‐instruments.com/)
Polyamide membrane filter (0.2 µm, Whatman)
Bottle‐top filter unit (Nalgene)
Oasis HLB column: 15 µm, 20 mm × 1.2 mm (Waters)
Allure Biphenyl column, 5 µm, 50 mm x 2.1 mm (Restek, http://www.restek.com)
4000 QTRAP triple quadrupole mass spectrometer equipped with a Turbo V source (AB Sciex)
Analyst 1.5.1 software (AB Sciex)
Prominence HPLC consisting of two HPLC pumps (LC‐20AB—binary, LC‐20AD—isocratic), an autosampler (SIL‐20AC), a column oven including a 6‐port switching valve (CTO‐20), and a controlling unit (CBM‐20A) (Shimadzu)

Additional reagents and equipment for collection of blood from the retro‐orbital sinus of mice (Rathkolb et al., ) and preparation of plasma ( protocol 2 )

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Figures

Figure 1. Equipment of the clinical chemistry laboratory at the GMC used for blood sample processing. (A ) Li‐heparin‐coated (orange cap) and EDTA‐coated (red cap) sample tubes used for blood collection for clinical‐chemical analyses, ELISAs, and other assays. (B ) Biofuge fresco (Heraeus) used to separate plasma from cells. (C ) Plasma samples for the measurement of clinical chemical parameters; 1.5‐ml sample tubes containing the plasma specimens fit into the bigger tubes delivered with the sample racks for the AU480‐analyzer.

View Image

Figure 2. Equipment of the clinical chemistry laboratory of the GMC used for the determination of clinical chemical parameters in mouse plasma. (A ) The AU480 clinical chemistry analyzer (Beckman Coulter). (B ) Racks with prepared samples are loaded into the sample supply of the analyzer to be processed. Here, the formerly used AU400 analyzer (Olympus) is shown.

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Figure 3. Principles of ELISA technology. (A ) Sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme‐linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form (e.g., color change into yellow). (B ) Competitive ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) enzyme‐conjugated antigen is added, unlabeled antigen from samples and the enzyme‐conjugated antigen compete for binding to the capture antibody; (4) substrate is added, and is converted by enzyme to detectable form (e.g., color change into yellow). Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color development.

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Figure 4. Representative chromatogram of measured steroids in mouse plasma (including internal standards).

View Image

Videos

Literature Cited

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Key References

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Clinical Chemistry and Other Laboratory Tests on Mouse Plasma or Serum

Abstract

Table of Contents

Materials

Basic Protocol 1: Serum Preparation from Mouse Blood

Basic Protocol 2: Plasma Preparation from Mouse Blood

Basic Protocol 3: Analysis of Clinical‐Chemical Parameters in Mouse Plasma Using the AU480 System

Basic Protocol 4: Measurement of Mouse Osteocalcin by a Commercially Available Sandwich ELISA (Direct Detection)

Basic Protocol 5: Measurement of Mouse/Rat PINP by a Commercial Available Competitive ELISA

Basic Protocol 6: Quantification of Steroids in Mouse Plasma Using Online SPE Coupled to LC‐MS/MS

Figures

Videos

Literature Cited