Alkaline Phosphatase for the Diagnostics and Life Sciences Industry

Table of Contents

Your Perfect Partner 3

Alkaline Phosphatase (AP) Product Overview 4

Alkaline Phosphatase for Immunology 5

Alkaline Phosphatase for ImmunologyCharacteristics of Recombinant and Native Alkaline Phosphatase 6

Alkaline Phosphatase for ImmunologyFeatures of Recombinant AP Conjugates 7

Alkaline Phosphatase for ImmunologyRecombinant AP Mutein: Inactive AP for Interference Elimination 8

Recombinant Alkaline Phosphatase for Molecular Diagnostics 9

References 10

Chromatography columns for the large scale production of biosubstances

Products are for further processing only.

3

Your Perfect Partner

Peeler centrifuge with automated drawdown for sealed separation of solids

Chromatography equipment for large-scale production of enzymes: Fraction containers

Roche Custom Biotech is ready to serve you in a long-term relationship as a quality-conscious, reliable partner for raw material supply.

Our experience in immunology and molecular diagnostics supports your diagnostic kit manufacturing, comprehensively and professionally, from the beginning of assay development to commercialization.

4

Alkaline Phosphatase (AP) Product Overview

The AP portfolio of Roche Custom Biotech comprises two AP product classes:

- Native AP from New Zealand bovine intestine (BSE-free)

- Recombinant AP produced in the yeast Pichia pastoris

The high quality of both of these product groups serves all your needs for immunology and molecular diagnostics. These AP products are the perfect building blocks for stable and reproducible AP conjugates.

AP Mutein, the inactive variant of the recombinant highly active AP, is also available from Roche Custom Biotech for applications involving AP interference elimination.

Properties of Alkaline Phosphatase

Nomenclature Orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1

Subunits Homodimer (Zn2+ is essential for activity)

Molecular weight (MW) 56 kD (by SDS-PAGE, monomer)

Inhibitors Inorganic phosphate, metal chelating agents, divalent heavy metal ions (e.g., Be2+, Zn2+), many amino acids (e.g., L-phenylalanine, L-tryptophane, L-cysteine), iodoacetamide

Activators Mg2+, Co2+, Mn2+

pH Optimum 9.8 (activity)

pH Optimum 8.0 (stability)

Specificity Alkaline Phosphatase catalyzes the hydrolysis of numerous phosphate esters, such as esters of primary and secondary alcohols, saccharides, cyclic alcohols, phenols and amines. Phosphodiesters do not react. The enzyme hydrolyzes inorganic pyro-phosphate. The kinetic properties of the enzyme depend on many factors, such as purity of enzyme, concentration of enzyme in the assay, buffer, pH etc.

Recombinant Alkaline Phosphatase, from ��Pichia pastoris for immuno-diagnostics:

EIA Grade, highly active -EIA Grade, highly active, carbohydrate-reduced -AP Mutein, inactive AP -

Alkaline Phosphatase, bovine intestine, �� EIA Grade

Recombinant Alkaline Phosphatase, �� Molecular Biology Grade

Substrates for the detection of AP conjugates:��pNPP for ELISAs -BCIP for blots and immunohistochemistry and -immunocytochemistry

5

Alkaline Phosphatase for Immunology

Native Alkaline Phosphatase, EIA GradeAP, EIA Grade is specially developed as a raw material for producing stable and highly repro ducible antibody- and antigen-enzyme conjugates. These AP conjugates are ideal for enzyme immunoassays, providing a wide range of detection options using colorimetric, fluorimetric or luminometric substrates.

Native AP, EIA Grade is:the standard for alkaline phosphatase conjugates��from bovine intestine sourced from New Zealand, ��a country acknowledged as being BSE-free

Recombinant Alkaline PhosphataseRecombinant highly active AP is the ideal alternative to conventional AP products derived from bovine intestine. No animal-derived components are used in the production process. Therefore the risk of Bovine Spongiform Encephalopathy (BSE) or other infections caused by animals is eliminated.

Recombinant AP of Roche Custom Biotech:uses the yeast, �� Pichia pastoris, as expression system.has a clearly defined molecular structure.��has a high specific activity: �� ≥7000 U/mg.is also available in a carbohydrate-reduced (CR) ��quality.

Highly active recombinant AP comprises fewer isoenzymes and has just slightly different N-glyco-sylation compared to native AP. This N-glycosylation difference is due to production in Pichia pastoris, which produces a slightly higher mannose branching pattern.An optional proprietary deglycosylation digests Recombinant AP’s mannose branching pattern. This treatment produces a highly active, carbo hydrate-reduced, recombinant AP with a carbo hydrate ratio comparable to that of native AP.

Model for N-glycosylation

For additional information please refer to references 2) and 3).

Native APAverage Carbohydrates:10%

Recombinant AP

16 – 22%

Recombinant AP, CR

6%

Asn – GN – GN – M – GN

M

M – M

M – GN

GN

––

––

Asn – GN – GN – M – GN

MM – M

M – GN GN

––

–

–

Asn – GN

Asn – GN

Asn – GN – GN – M

M – M M – M ––

M M

[M]0-2

M – M [M]0-2–

–

–

–

–

Asn – GN – GN – M

M

– M

M – M

––

M

M

[M

]0-2

M

– M

[M]0-2

–

–––

–

Asn: AsparagineGN: GlcNAcM: Mannose

Model 3D structure of the homo dimer of recombinant highly active AP

� KK�� AP subunits � � �Catalytic active Ser 92: AP Mutein recombinant is created

by the single point mutation Ser 92 ⟶ Ala 92 which yields an inactive enzyme.

� � ��Asn residues: Potential glycosylation sites ��� Lys residues

6

Alkaline Phosphatase for Immunology Characteristics of Recombinant and Native Alkaline Phosphatase

Physical data

Native AP, EIA Grade

Recombinant AP, highly active

Recombinant AP, highly active, CR

Catalog Number 10 556 602 103 03 137 031 103 03 535 452 103

IEP (IEF, CE) 5.8 – 6.6 3.6 – 4.7 5.2 – 6.0

MALDI-TOF MS Total molecular mass Molecular mass protein Molecular mass carbohydrates

116 kD 104 kD (= 90%) 12 kD (= 10%)

124 ± 10 kD104 kD (= 84%) 20 ± 10 kD (16 ± 6%)

111 kD 104 kD (= 94%) 6.5 kD (= 6%)

Accessible N-glycosylation sites Branching type

2/subunit high branched type (hybrid type) GlcNAc, Mannose, no NeuAc detected

2/subunit higher branched type (hybrid type) GlcNAc, Mannose, no NeuAc detected

2/subunit reduced branched type

GlcNAc, Mannose,no NeuAc detected

O-glycosylation sites Not detected Not detected Not detected

Number of isoenzymes present based on protein

7 (IEF-2D) 5 (IEF-2D)

3 (MS) 1 (MS)

1 (MS) 1 (MS)

Specifications

Native AP, EIA Grade

Recombinant AP, highly active

Recombinant AP, highly active, CR

Catalog Number 10 556 602 103 03 137 031 103 03 535 452 103

Appearance Clear colorless solution in NaCl, 3 mol/l; ZnCl2, 0.1 mmol/l; TEA, 30 mmol/l;MgCl2, 1 mmol/l

Clear colorless solution in NaCl, 3 mol/l; ZnCl2, 0.1 mmol/l; TEA, 30 mmol/l;MgCl2, 5 mmol/l

Clear colorless solution in NaCl, 3 mol/l; ZnCl2, 0.1 mmol/l; TEA, 30 mmol/l;MgCl2, 5 mmol/l

pH Value 7.0 – 8.0 7.0 – 8.0 7.0 – 8.0

Protein(A280: 1 mg/ml = 1;against H2O) 10 mg/ml 20 ± 1 mg/ml 20 ± 1 mg/ml

Specific activity(37°C; pNPP) 3000 U/mg 7000 U/mg 7000 U/mg

Purity (HPLC) 95% 95% 95%

Amino groups 8 – 16 moles/mole 5 – 13 moles/mole 5 – 13 moles/mole

Carbohydrates No limit No limit 7%

Stability +2 to +8°C 15 months +2 to +8°C 12 months +2 to +8°C 12 months

7

Alkaline Phosphatase for ImmunologyFeatures of Recombinant AP Conjugates

Conjugation experiments show that highly active, recombinant Alkaline Phosphatase, EIA Grade is the ideal replacement for conventional alkaline phosphatase products.

Recombinant AP conjugates show:similar chromatographic profi les compared to ��conventional AP productshigh reproducibility and lot-to-lot consistency of ��conjugates (see stability data below)high sensitivity in ELISAs��

Stability of < DIG > -AP conjugates with rec. AP, highly active, EIA Grade (2 lots)and bovine AP, highly active, EIA Grade (1 lot)

Conjugation of AP with PAB<DIG>S-Fab(IS) via Nakane (APox): Recombinant, highly active AP displays high stability, which is nearly identical to the native enzyme. In Roche's model system, Fab<DIG>-AP conjugates exhibit good repro ducibility and lot-to-lot consistency.

at 4°C

Rec

over

y of

enz

yme

activ

ity [

%]

at 35°C

Rec

over

y of

enz

yme

activ

ity [

%]

100

80

60

40

20

0

weeks 0 2 4 6 12

weeks 0 2 4 6 12

100

80

60

40

20

0

8

Recombinant AP Mutein is the inactive form of the highly active Recombinant AP. AP Mutein eliminates interference directed against the active AP. The inactivating moiety in AP Mutein is a single point mutation located in the AP active site.

Recombinant AP Mutein is the optimal choice for reducing AP-related interference in your applications.

Alkaline Phosphatase for Immunology Recombinant AP Mutein: Inactive AP for Interference Elimination

Recombinant AP Mutein

Catalog Number 04 781 007 103

Appearance White to yellowish lyophilizate

pH Value 7.0 – 8.0

Protein (A280; 1 mg/ml = 1; against H20) 0.2 mg protein/mg Iyophilizate

Specific activity (37°C; pNPP) 10 U/mg protein

Stability +2 to +8°C 24 months

Specifications

Recombinant AP Mutein:is equivalent to highly active Recombinant AP ��with the exception of a single inactivating point mutation.is produced using the same initial production ��steps as the active enzyme in Pichia pastoris.production involves the same deglycosylation ��step as established for highly active AP, CR.has a specific activity of �� 10 U/mg.is provided in EIA Grade quality.��

Recombinant AP, Mol Biol Grade 1 U/µl

Recombinant AP, Mol Biol Grade 20 U/µl

Catalog Number 04 571 550 103 04 571 363 103

Appearance Clear colorless solution in Tris-HCI, 25 mmol/l; MgCl2, 1 mmol/l; ZnCl2, 0.1 mmol/l; glycerol, 50% (v/v); pH approximately 7.6 at 4°C

Volume activity 1 x 103 U/ml 20 x 103 U/ml

Specific activity 5 x 103 U/mg 5 x 103 U/mg

Endonucleases Not detectable (100 U/4 h/37°C) Not detectable (100 U/4 h/37°C)

Exonucleases Not detectable (60 U/4 h/37°C) Not detectable (60 U/4 h/37°C)

RNases Not detectable (100 U/1 h/37°C) Not detectable (100 U/1 h/37°C)

Nicking activity Not detectable (100 U/4 h/37°C) Not detectable (100 U/4 h/37°C)

5'-Labeling Typical incorporation rate 30% Typical incorporation rate 30%

Stability -15 to -25°C 24 months -15 to -25°C 24 months

Specifications

Recombinant Alkaline Phosphatase for Molecular Diagnostics

Recombinant Alkaline Phosphatase, Molecular Biology Grade meets all your needs for applications in molecular biology and diagnostics. Additional production steps ensure the removal of DNases, RNases, nicking activity and exonucleases, which can lead to the degradation of DNA and RNA.

In addition to its consistent quality and stability, the major advantage of Recombinant Alkaline Phosphatase, Molecular Biology Grade, is its simple inactivation by heating at +75°C for 5 minutes.

The entire workflow, including restriction enzyme digestion, dephosphorylation, enzyme inactivation, ligation or 5' end-labeling, can be performed in a single tube.

Recombinant AP, Mol. Biol. Grade, is especially suitable for dephosphorylation of 5' phosphorylated ends of DNA and RNA. Dephosphorylated vector DNA is prevented from self-annealing prior to the insertion of DNA fragments. The special quality of Recombinant AP, Molecular Biology Grade avoids the degradation of probe-enzyme conjugates used to detect amplified and non-amplified DNA/RNA in probe detection assays.

9

10

1) Genetic Complexity, Structure and Characterisation of Highly Active Bovine Intestinal Alkaline Phosphatases, JBC, 273, 36, 23353 – 23360 (1998), Th. Manes, M.F. Hoylaerts, R. Mueller, F. Lottspeich, W. Hoelke, J.L. Millán

2) Glycosylation of Pichia Pastoris derived Proteins, Biotechnol. Appl. Biochem. 30, 193 – 200 (1999), R.K. Bretthauer, F.J. Castellino

3) Heterologous Protein Expression in the Methylotropic Yeast Pichia Pastoris FEMS Microbiology Reviews 24, 45 – 66 (2000), I. Ceveghino, I. Gregg

4) Heterologous Protein Production in Methylotropic Yeasts, Appl. Microbiol. Biotechnol. 54, 741 – 750 (2000), G. Gellissen

5) Detection of attograms of antigen by a high-sensitivity enzyme-linked immunoabsorbent assay (HS-ELISA) using a fluorogenic substrate, J. Immunol. Meth. 38, 125 (1980), A. Shalev, A. H. Greenberg and P. McAlpine

6) Which of the commonly used marker enzymes gives the best results in colorimetric and fluorometric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or b-galactosidase? J. Immunol. Meth. 79, 27 (1985), B. Porstmann, T. Porstmann, E. Nugel and U. Evers

Products are for further processing only.

References

11

Customers in Europe, Middle East, Africa, and Latin America:Roche Diagnostics Deutschland GmbHSandhofer Straße 11668305 Mannheim, GermanyPhone +49 621 759 8580Fax +49 621 759 8610

Customers in the United States:Roche Diagnostics CorporationRoche Applied Science9115 Hague RoadP.O. Box 50414Indianapolis, IN 46250-0414, USAPhone +1 800 428 5433, ext. 14649 (toll-free)Fax +1 317 521 4065

Customers in Canada:Roche Diagnostics201, Boulevard Armand-FrappierHV 4A2 Laval, Québec, CanadaPhone +1 450 686 7050Fax +1 450 686 7012

Customers in Japan:Roche Diagnostics K.K.Roche Custom BiotechApplied Science6-1, Shiba 2-chomeMinato-ku, Tokyo 105-0014, JapanPhone +81 3 5443 5285Fax +81 3 5443 7934

Customers in Asia Pacific:Roche Diagnostics Asia Pacific Pte. Ltd.Regional Sales and Market Development298 Tiong Bahru Road# 16-01/06 Central PlazaSingapore, 168730Phone +65 6371 6638Fax +65 6371 6601

Your Roche Custom Biotech Customer Service

custombiotech.roche.com

Published by

Roche Diagnostics GmbHSandhofer Straße 11668305 MannheimGermany

© 2010 Roche Diagnostics.All rights reserved.

05861608990 1110