Boehringer Mannheim Gmbh

Karl Wulff

Fritz Stahler

Kurt W Naujoks

Hans-Georg Batz

Helmut Lenz

Wolfgang Gruber

Winfried Albert

Christa Hubner-Parajsz

August W Wahlefeld

Herbert Jungfer

Hans-Ralf Linke

Gerhard Michal

Martin Gerber

Willie Gerhardt

Karl-Dietrich Gundermann

Fritz Staehler

Pancreatic .alpha.-amylase is determined in body fluid in the presence of salivary .alpha.-amylase by combining the body fluid with a monoclonal antibody that inhibits salivary .alpha.-amylase by 95% or more and inhibits pancreatic .alpha.-amylase by 50% or less, and then detecting pancreatic .alpha.-amylase with a system for the detection of .alpha.-amylase. The monoclonal antibody may be in immobilized form. The monoclonal antibody is preferably produced by immunizing a Balb/c mouse or an AJ mouse with a specific immunogen for a specific number of times, fusing .beta.-lymphocytes obtained from the mouse with a myeloma cell line to form an antibody producing hybridoma cell line, cloning and screening the hybridoma cell line for the desired monoclonal antibody, and isolating the monoclonal antibody. The immunogen contains modified or unmodified salivary .alpha.-amylase, aluminum hydroxide and Bortadella pertussis.

Process and monoclonal antibody for the specific determination of

In body fluids containing saliva alpha amylase and pancreatic alpha amylase, pancreatic alpha amylase is determined with a monoclonal antibody which specifically binds but does not inhibit saliva alpha amylase and which has a cross-reactivity of 5% or less toward pancreatic alpha amylase. The monoclonal antibody binds salvia alpha amylase to form a complex which is separated to permit determining pancreatic alpha amylase with an amylase detection system. The complex may be separated by precipitating with a precipitating agent such as an anti-antibody or protein A, or by immobilizing the monoclonal antibody on a solid carrier. The monoclonal antibody is preferably produced by immunizing a host animal with an immunogen containing aluminum hydroxide, Bordatella pertussis and native saliva alpha amylase at least 7 times over a period of at least 9 months, fusing B lymphocytes of the immunized host animal with a transforming agent to produce hybridomas, cloning and culturing the hybridomas, and screening antibodies formed.

The present invention provides a process for the preparation of compounds of the general formula: ##STR1## in which X is a hydroxyl group and X.sub.1 is a hydrogen atom or a hydroxyl group, and especially of luciferin, by the reaction of D-cysteine with 2-cyanomono- or -dihydroxybenzothiazole obtained from 2-chloromono- or -dimethoxybenzothiazole via a demethylation, wherein the demethylation is accomplished by reaction with iodotrimethylsilane or with a mixture of phenyltrimethylsilane and iodine and hydrolysis under mild conditions of the silyl derivative formed.

Process for the preparation of luciferin compounds

The present invention provides a method for the determination of peroxidase in the presence of a compound, the enzyme-catalysed oxidation of which with a peroxy compound results in the emission of light, which is measured, wherein 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide of the formula ##STR1## is used as light-emitting compound. The present invention also provides a reagent for the determination of peroxidase, comprising 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide, a system providing hydrogen peroxide and a buffer substance of pH 6 to 9.

Reagent and method for the determination of peroxidase

Creatine kinase is determined by the reaction of creatine phosphate with adenosine diphosphate with the formation of adenosine triphosphate, reaction of the latter with luciferin and oxygen in the presence of luciferase and diadenosine pentaphosphate with the formation of oxyluciferin and adenosine monophosphate, and measurement of the light thereupon emitted, the reaction being performed with a saturated concentration of adenosine diphosphate and substrate, in the presence of 1 to 10 millimoles per liter of AMP at pH values of 5.8 to 7.5, with at least 50 units of luciferase per test. It is desirable to operate in the presence of diadenosine pentaphosphate or sodium fluoride and a sequestering agent.

Method and reagent for the determination of creatine kinase

A method for the quantitative determination of an oxidized pyridine co-enzyme which comprises reacting a sample containing oxidized pyridine co-enzyme in the presence of its specific alcohol dehydrogenase with an aliphatic alcohol of 8 to 16 carbon atoms, oxidizing the aldehyde formed with reduced flavine mononucleotide and oxygen in the presence of luciferase and of the particular FMN-reductase which is independent of the co-enzyme to be determined and of the reduced substrate co-enzyme of the latter, and measuring the resulting light emission as a measure of the initial oxidized pyridine co-enzyme content.

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Weilheim, Germany

Karl Wulff