Chemistry: analytical and immunological testing – Metal or metal containing – Present in biological fluids
Reexamination Certificate
1998-05-27
2001-11-27
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Metal or metal containing
Present in biological fluids
C436S073000, C436S077000, C436S166000
Reexamination Certificate
active
06323036
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to reagents and methods for releasing and measuring lead in blood.
2. Background Discussion
Lead and its compounds have become widely distributed in the environment over the centuries. Lead is known to be toxic and have deleterious effects on humans. The toxicity of lead is well documented. Trace amounts of lead can adversely affect the function of various organs of the human body, especially in small children. It is now generally recognized that lead poisoning occurs at blood levels as low as 10 to 15 &mgr;g/dL. Thus, the detection and measurement of lead in blood is extremely important in providing universal blood screening and in monitoring occupational lead exposure.
Most lead screening programs use atomic absorption or anodic stripping voltametry methods for the determination of trace amounts of lead. Both methods are cumbersome to use, susceptible to contamination, and costly. Enzyme-based biodetection systems of lead, which employ &dgr;-aminolevulinic acid dehydrase (ALAD) and isocitrate dehydrogenase, have also been proposed. An automated fluorimetric lead assay, which uses inhibition of ALAD by lead is disclosed in PTC International Publication No. WO95/17679 to long et al., which discloses the detection of lead in a sample by adding a lead recovery agent to an assay solution containing lead, adding a disulfide enzyme to the assay solution and correlating the activity of the disulfide enzyme to the amount of lead in the sample.
The Wong et al. reference discloses the use of acids such as trichloroacetic acid, nitric acid, 5-sulfosalicylic acid, or perchloric acid as an initial treatment step in separating lead ions from a blood sample. However, the resulting lead-containing aqueous supernatant must be neutralized with a buffer containing a lead recovery agent before analysis. Otherwise, the lead is unavailable for measurement.
Other methods of sample pretreatment include acid digestion or dilution of blood with a matrix modifier such as a nitric acid solution of ammonium phosphate and a nonionic surfactant, prior to analysis by atomic absorption.
SUMMARY OF THE INVENTION
The present invention relates to the release and measurement of the lead content of a biological matrix such as whole blood, with anticoagulants such as heparin or ethylene-diamine tetraacetic acid (EDTA). A releasing reagent is used to extract the lead content in the form of lead ions from the biological matrix into a supernatant liquid, and comprises an acid solution capable of releasing lead ions from the biological matrix, and at least one or more salts capable of solubilizing the released lead ions from the biological matrix.
When EDTA is used as the anticoagulant, an EDTA complexing ion is used to block the released lead ions from complexing with EDTA and thereby prevent complexation of the released lead ions by EDTA.
The invention also relates to a calorimetric reagent that is used to determine the amount of released lead ions, comprising porphyrin and a quaternary ammonium salt.
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Chapoteau Eddy
Craine Jonathan
Czech Bronislaw P.
Bayer Corporation
Klawitter, Esq. Andrew L.
Rodman & Rodman
Soderquist Arlen
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