This document provides a pipetting scheme and instructions for performing an assay to measure acid phosphatase levels using a reagent kit. The assay involves hydrolyzing 1-naphthyl phosphate with acid phosphatase to form phosphate and 1-naphthol, which is then converted to an azo dye for measurement. The increase in absorbance at 405nm over time is proportional to the acid phosphatase activity in the sample. The document provides details on sample preparation, reagent preparation, controls, reference values and automation options.
This document provides a pipetting scheme and instructions for performing an assay to measure acid phosphatase levels using a reagent kit. The assay involves hydrolyzing 1-naphthyl phosphate with acid phosphatase to form phosphate and 1-naphthol, which is then converted to an azo dye for measurement. The increase in absorbance at 405nm over time is proportional to the acid phosphatase activity in the sample. The document provides details on sample preparation, reagent preparation, controls, reference values and automation options.
This document provides a pipetting scheme and instructions for performing an assay to measure acid phosphatase levels using a reagent kit. The assay involves hydrolyzing 1-naphthyl phosphate with acid phosphatase to form phosphate and 1-naphthol, which is then converted to an azo dye for measurement. The increase in absorbance at 405nm over time is proportional to the acid phosphatase activity in the sample. The document provides details on sample preparation, reagent preparation, controls, reference values and automation options.
This document provides a pipetting scheme and instructions for performing an assay to measure acid phosphatase levels using a reagent kit. The assay involves hydrolyzing 1-naphthyl phosphate with acid phosphatase to form phosphate and 1-naphthol, which is then converted to an azo dye for measurement. The increase in absorbance at 405nm over time is proportional to the acid phosphatase activity in the sample. The document provides details on sample preparation, reagent preparation, controls, reference values and automation options.
Warm working reagents and cuvettes up to the desired temperature.
Orthophosphoric - Monoester Phospho- Temperature must be kept constant (±0.5°C) for the duration of the test. hydrolase Assay temperature 25°C / 30°C / 37°C Assay Semi micro Macro Package Size [REF] 10660 6 x 15 ml Complete test kit Sample 100 µl 200 µl [IVD] Working reagent 1000 µl 2000 µl Mix, read the absorbance A1 after 5 minutes and start the stop watch Method1 at the same time. Read the absorbance A2 exactly after 3 minutes at 1-Naphthyl phosphate is hydrolysed by acid phosphatase (Ac. P.) to phos- 30°C and 37°C or after 5 minutes at 25°C. A2 – A1 = A phate and 1-naphthol, which is converted with FRTR-salt (see "Contents", [SUB]) to an azo dye. The increase of absorbance at 405 nm is propor- Calculation tional to the total acid phosphatase activity in the sample. Calculate the total acid phosphatase activity in the sample using the following factors: Reaction Principle Ac. P. U/l Semi micro / Macro assay 1-Naphthyl phosphate + H2O phosphate + 1-naphthol Assay temperature 25°C 30°C or 37°C 1-Naphthol + FRTR-salt azo dye Total acid phosphatase A x 149 248 Contents Conversion factor of the traditional units (U/l) in SI-units (kat/l): [BUF] 1 x 100 ml Buffer Solution Citrate buffer (pH 5.2) 100 mmol/l 1 U/l = 16.67 x 10-3 µkat; 1 µkat/l = 60 U/l [SUB] 6 x 15 ml Substrate (powder) Performance Characteristics 1-Naphthyl phosphate 143 µmol Linearity FRTR-salt 15 µmol If the absorbance change exceeds A = 0.3 at 30°C or 37°C or A = 0.5 at (4-chloro-2-methylphenyl diazonium salt) 25°C or if the activity is higher than 63 U/l dilute 0.1 ml of sample with 0.2 ml physiological saline (0.9%) and repeat the assay using this dilution. [STAB] 1 x 4 ml Stabiliser Multiply the result by 3. Acetic acid 0.7 mmol/l Typical performance data can be found in the Verification Report, acces- sible via: Additional material recommended but not supplied with the kit www.human.de/data/gb/vr/en-acp.pdf or [REF] 13160 [CAL] 4x AUTOCAL www.human-de.com/data/gb/vr/en-acp.pdf for 5 ml lyophilised calibrator for HUMAN Clinical Chemistry Reference Values2 Reagents Total acid phosphatase [REF] 13511 13512 13951 13151 Assay temperature 25°C 30°C 37°C [CONTROL] HumaTrol N HumaTrol P SERODOS SERODOS plus 6 x for 5 ml 6 x for 5 ml 6 x for 5 ml 6 x for 5 ml Men up to (U/l) 3.6 5.0 6.5 Control serum Control serum Control serum Control serum Women up to (U/l) 3.0 4.2 5.5 normal abnormal normal abnormal lyophilised control serum for HUMAN Clinical Chemistry Quality Control Reagents For quality control use the control material recommended in “Additional material recommended but not supplied with the kit” or other suitable Reagent Preparation control material. The control intervals and limits must be adapted to the Working reagent (total acid phosphatase determination) individual laboratory requirements. Values obtained should fall within Dissolve the contents of one bottle [SUB] with exactly 15 ml of [BUF]. established limits. Each laboratory should establish corrective measures Mark label with date of preparation. to be taken if values fall outside the limits.
Reagent Stability Automation
The reagents are stable up to the stated expiry date when sealed and Proposals to apply the reagents on analyzers are available on request. stored at 2...8°C. Each laboratory has to validate the application in its own responsibility. After reconstitution, the working reagent is stable for 5 days at 2...8°C References and for 24 hours at 15...25°C protected from light. 1. Hillmann G., Z. Klin. Chem. Klin. Biochem. 9, 273 (1971) Specimen 2. Junge W. et al., Data presented at the 45th National Meeting of the Serum, no plasma! AACC, New York, July 1993 Avoid hemolytic and icteric sera! Sample Preparation EN-ACP INF 1066001 GB 02-2011-14 | Stabilise samples, AUTOCAL and controls by addition of one drop of [STAB] to 1 ml sample immediately after separation / reconstitution. Thus samples remain stable for 3 days at 2...8°C and 24 hours at 15...25°C. Assay Wavelength: Hg 405 nm Optical path: 1 cm Temperature: 25°C, 30°C or 37°C Measurement: against air (increasing absorbance)
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