Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/827—Actinoplanes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/886—Streptomyces
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/886—Streptomyces
  • Y10S435/893—Streptomyces chartreusis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
  • Y10S435/913—Aspergillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
  • Y10S435/921—Candida
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
  • Y10S435/921—Candida
  • Y10S435/922—Candida albicans
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
  • Y10S435/933—Penicillium

Definitions

• ABSTRACT Total cholesterol or bound cholesterol in a sample is determined by treating the sample with cholesterol esterase, thereby releasing the bound cholesterol, and then determining the resulting total cholesterol by known methods; specifically preferred are cholesterol esterases from Candida rugosa ATCC 14830, Rhizopus spec. WS 90027 and Aspergillus spec. WS 90030.
• the present invention relates to a method of determining cholesterol, and more specifically to a method of determining either total cholesterol or bound cholesterol.
• Cholesterol is present in biological matter, such as serum or the like, partially in free form and partially in bound form as ester.
• biological matter such as serum or the like
• the released cholesterol can then be determined either chemically or enzymatically by one of the known methods.
• the chemical determination may be performed, for example, by the Lieberrnann-Burchard method, and enzymatic determination may be performed by means of cholesterol oxidase, cholesterol dehydrogenase or cholesterol dehydrase. Since the individual cholesterol esters as well as the free cholesterol are known to have different extinction coefficients in the chemical methods of determination, it is necessary to transform the cholesterol esters to free cholesterol by alkaline hydrolysis.
• the alkaline saponification of the bound cholesterol is a troublesome and time-consuming step in the procedure.
• the relatively agressive reagents used may lead to a decomposition of the cholesterol.
• a hydrolysis must be performed under relatively mild conditions, and this in turn undesirably increases the length of time required for the determination.
• the alkaline liberation of the cholesterol is especially disadvantageous when the determination of the cholesterol is afterwards to be performed by the preferred enzymatic methods. Since the enzymes are inactivated, as it is known, in the strongly alkaline medium, the hydrolyzate must be acidified by the addition of acid to pH 5 to 8 before the enzymatic determination can be started. All this results in the fact that the determination of the total cholesterol or of the bound cholesterol still takes an undesirably long time and requires too much work.
• the present invention provides a process for the determination of total cholesterol or bound cholesterol which substantially obviates the above-mentioned disadvantages.
• the process of the invention comprises the determination of total cholesterol or of bound cholesterol by releasing the bound cholesterol using cholesterol esterase, followed by determination of the released cholesterol by known methods.
• cholesterol esterases are capable of cleaving quantitatively, within a very short time, all of the cholesterol esters that occur. This is especially surprising also because in the known cholesterol esterases there are considerable differences with regard to their activity against various cholesterol esters.
• Cholesterol esterases from microorganisms have proven to be especially suited for the process of the invention. They have proven superior to cholesterol esterases of other origin in regard to speed of cleavage and effectiveness and they are therefore preferred within the scope of the invention.
• Candida rugosa also referred to as Cylindracea
• WS 90031 a cholesterol esterase derived from Candida rugosa
• Aspergillus spec. WS 90030 a cholesterol esterase derived from Candida rugosa (also referred to as Cylindracea) ATCC 14830 and WS 90031, respectively, and from Aspergillus spec. WS 90030.
• These two microorganisms may be used directly as such or in processed form, e.g., in the form of an acetone dry powder, within the scope of the invention.
• a concentrated cholesterol esterase preparation made from these microorganisms, there being a special advantage in the fact that a certain concentration may in this case be achieved very simply.
• Candida rugosa is a microorganism that is produced on a large technical scale and is available commercially.
• the customary commercial form is an acetone dry powder stabilized with lactose, which has proven to be outstandingly suited for the invention.
• Actinom yces aureoverticillium WS 90002 Acn'nam yces cyaneofurcatus WS 90003 Acu'nomyces griseomycini WS 90004 Am'nomyces longisparus-fl.
• WS 90005 Acn'nomyces malachiticu: WS 90006
• Actinomyces toxym'cini WS 90008 Aclinomyces variabih's WS 90009 Srrepwmyces .rpec.
• WS 90010 Srreplomyces autotrophicus WS 9001 l Streptomyccs canes-certs WS 90012 Streptomyces charlreusis WS 90013 Streptomyces michiganensis WS 90014 Streptomyces murinu: WS 90015 Streptomyces hachijoensis WS 90016 Streptomyces caelestes WS 90017 Streptomyces tendae WS 900l8 Nocardia rubra WS 90019 Candida m ycoderma WS 90020 Candida albicans WS 9002] Candida albicans WS 90022 Candida albicans WS 90023 Candida spec.
• WS 90024 Cunninghamella elegans WS 90025 Mucar mucedo WS 90026 Rhizopus spec. WS 90027 Penicillium spec. WS 9002B Aspergillus spec. WS 90029
• cholesterol esterases of other origin may also be used in many cases.
• an especially important advantage of the process of the invention consists in the fact that it makes possible an all-enzymatic determination of total cholesterol. It is important in this case that, with the preferred cholesterol esterase preparations made from microorganisms; a rapid and quantitative release of the cholesterol from its esters is possible. Especially with the preferred microorganisms mentioned above, it is possible by the direct addition of same in a very small quantity, with the maintenance of the pH values and temperatures which are desirable in the subsequent enzymatic determination of cholesterol, to achieve within a few minutes a quantitative release of the cholesterol, it having been found that the common carbohydrate-based stabilizing agents which are used for such microorganisms do not interfere with the cholesterol determination performed within the framework of the allenzymatic process.
• a separated and concentrated cholesterol esterase preferably one obtained from microorganism, may also be used for the process of the invention.
• a suitable concentration may be achieved by setting out from an acetone dry powder of the microorganism or other biological material and subjecting it to a dialysis, a treatment with weakly basic anion exchanger and to an ammonium sulfate fractionation. In this manner, it is easy to achieve a -fold to 30-fold concentration of the cholesterol esterase.
• a preparation on a carbohydrate basis, modified with diethylaminoethanol groups, has proven to be an especially suitable weakly basic anion exchanger.
• the fraction between 1.8 and 2.4 moles of ammonium sulfate is preferably obtained.
• the enzyme fraction thus obtained is then chromatographed, preferably on the above-named exchanger material.
• microorganisms which have been cultivated in a nutrient medium containing cholesterol ester.
• the cholesterol ester or a mixture of cholesterol esters may be added during the cultivation as the sole source of carbon, or may be used together with another carbon source.
• microorganisms which are obtained in a multi-stage cultivating process, in which they are cultivated in the first stage on a suitable carbon supplier, such as glycerin, and in the second stage on a cholesterol ester.
• a suitable cultivation process is described, for example, in German Published Specifications (Offenlegungsschriften") Nos. 2,224,133 and 2,307,518.
• the cholesterol esterase from Candida rugosa ATCC l4830 used preferentially in accordance with the invention has very good stability in the weakly acid region between pH 5 and 6.5.
• the optimum pH for the enzyme is 7.5.
• One peculiarity of the enzymes is that the catalytic reaction takes place especially well when the salt content of the reaction medium is relatively high.
• the process is performed in an 0.2 to 0.8 molar buffer solution.
• the pH may range between 4.5 and 7.5, and will preferably range, as stated above, between pH 5 and 6.5.
• the effectiveness of the cholesterol esterase is preferably increased by the addition of surface active substances. Especially preferred is the addition of hydroxypolyethoxydodecane.
• the process of the invention be performed allenzymatically, i.e., the cholesterol determination that follows is also performed enzymatically, preferably with the use of cholesterol oxidase.
• cholesterol dehydrase or dehydrogenase may also be used.
• Determination with cholesterol oxidase is described, for example, in German Offenlegungsschrift No. 2,224,132.
• the process therein described may advantageously be combined with the process of the invention.
• the determination of the oxygen consumption may be performed, for example, by gas chromatography or polarometry, or by the polarization method. These methods of determination are in the prior art.
• the hydrogen peroxide that forms may be determined titrimetrically, potentiometrically, and colorimetrically, as well as enzymatically.
• Enzymatic determination is preferred, with the use of catalase or peroxidase, especially determination by catalase in the presence of beta diketones such as acetylacetone, low alcohols and buffer containing ammonium ions, or determination by peroxidase in the presence of a chromogen such as 2,2-aminobenzothiazolinesulfonic acid.
• beta diketones such as acetylacetone, low alcohols and buffer containing ammonium ions
• peroxidase in the presence of a chromogen such as 2,2-aminobenzothiazolinesulfonic acid.
• Cholestenone is determined by means of keto reagents such as 2,4-dinitrophenylhydrazine, or by photometry at 240 lf the all-enzymatic determination of the total or bound cholesterol is performed with cholesterol oxidase, a cholesterol oxidase obtained from Nocardia erylhropolis ATCC l7895, Nocardia erythropolis ATCC 4277, Nocardia formica ATCC l48ll or Proacu'nomyces eryzhropolis NClB 9158 is preferably used.
• a reagent for the determination of cholesterol which consists of cholesterol esterase and a reagent for the determination of free cholesterol.
• a reagent of this sort consists of a cholesterol esterase of microbiological origin, cholesterol, oxidase, a system for the determination of hydrogen peroxide, or a system for the determination of cholestenone.
• a reagent in which the cholesterol esterase is one of the microorganisms mentioned further above, especially in the form of an acetone dry powder or a protein fraction obtained therefrom having cholesterol esterase activity.
• such a reagent consists of cholesterol oxidase, a cholesterol esterase preparation made from one of the above-mew tioned microorganisms, catalase, acetyl acetone, methanol and aluminum ion-containing buffer, individually or mixed.
• the reagent consists of cholesterol oxidase, a preparation made of the above-mentioned microorganisms having cholesterol esterase activity, peroxidase, chromogen and buffer, individually or mixed. 2,2'-aminobenzothiazolinesulfonic acid is preferred as the chromogen.
• the reagent of the invention consists of cholesterol oxidase, a cholesterol esterase preparation made from one of the named microorganisms, and a hydrazine derivative reacting with keto groups with the formation of hydrazone, and in some cases a buffer. 2,4-dinitrophenylhydrazine is preferred as the hydrazine derivative.
• the above-mentioned preferred reagent combinations may contain, in addition to the specified essential components, commonly used solvents, stabilizers and- /or suface active substances. All these additive substances are known to persons skilled in the art and commonly used in detection systems for hydrogen peroxide and cholestenone.
• the above-mentioned reagent combinations will contain the essential components in the following rations:
• microorganism cholesterol esterase 2 X to 5 X 10 units of catalase, 0.05 to 0.2 ml of acetyl acetone and 2 to 10 ml of methanol in 100 ml of a pH 5 to 7 buffer containing ammonium ions, plus, if desired, 0.02 to 0.3 ml of a surface active agent, preferably hydroxypolyethoxydodecane.
• a surface active agent preferably hydroxypolyethoxydodecane.
• microorganism cholesterol esterase and, if desired, 0.1 to 2.0 ml of surface active agent (preferably hydroxypolethoxydodecane), in 50 ml of pH 5 to 9 buffer, preferably 0.5 m of sodium phosphate pH 7.5 buffer.
• surface active agent preferably hydroxypolethoxydodecane
• EXAMPLE 2 To concentrate the cholesterol esterase activity, commercial acetone dry powder of Candida rugosa ATCC l4830 was dissolved in potassium phosphate buffer pH 6.0 and dialyzed against the same buffer. After removal of the lactose contained in the solution as stabilizer, the specific cholesterol esterase activity was 0.3 U per mg of protein in the dialyzed solution.
• the solution thus obtained was subjected to an ammonium sulfate fractionation.
• the protein fraction that precipitated between 1.8 and 2.4 M of ammonium sulfate was separated, and had a specific cholesterol esterase activity of 2.5 U/mg.
• the product obtained was again dissolved in pH 6.0 phosphate buffer, dialyzed against the same buffer until salt-free, and then chromatographed on a column filled with the same anion exchanger as above. Elution was again performed with 0.2 M of pH 6.0 phosphate buffer. A specific activity of 7 U per mg of protein was found in the fraction having cholesterol esterase activity.
• the concentrated cholesterol esterase preparation thus obtained was used in the cholesterol determination as described in Example 1, except that the amount used was only 0.001 mg with reference to protein. The results were the same as in Example 1.
• the cholesterol esterase from Candida rugosa can be further purified by the conventional methods of enzyme refinement.
• concentration procedures such as precipitation or fractionation with polyethyleneimine, organic solvents or salts, by chromatography through molecular sieve materials or weak anion exchanger with functional groups other than diethylaminoethanol groups. by protamine sulfate precipitation and the like.
• EXAMPLE 3 To 0.5 ml of serum in the one case and cholesterol standard in the other, I .0 ml of 0.5 M potassium phosphate pH 7.5 buffer containing 0.4% hydroxypolethoxydodecane, and 2.5 U of cholesterol esterase from Example 2 were added. This reaction mixture was incubated for 40 minutes at 37C. Then 0.25 ml of this solution was added to 3 ml of cholesterol reagent containing two parts acetic acid, three parts acetic acid anhydride and one part sulfuric acid (Liebermann-Burchardt reagent).
• EXAMPLE 4 l ml of 0.5 M potassium phosphate buffer containing 0.4% hydroxypolyethoxydodecane, and 0.2 U of the cholesterol esterase of Example 2, were added to 0.02 ml of serum. The reaction solution was incubated for 60 minutes 37C. Then the extinction (E at 240 nm was read in a suitable spectral photometer and the reaction was started with 0.1 U of sterol dehydrase obtained from Brevibaclerium sterolicum. After fifteen minutes, the extinction (E was again read. The concentration of the A cholestenone and hence of the cholesterol was found from the difference between the first and second reading on the basis of the molar extinction coefficient for A cholestenone at 240 nm. Measurement of a typical specimen gave 183 mg% total cholesterol.
• EXAMPLE 5 g of diammonium hydrogen phosphate was dissolved in 100 ml of water and adjusted to pH 7.0 with 85% phosphoric acid. Then 10 units of catalase were added. The solution thus obtained was added to a mixture of 0.2 ml of acetyl acetone, l0 ml of methanol and 0.1 g of hydroxypolyethoxydodecane to produce a volume of 100 ml. To this solution, 2.5 units of cholesterol esterase from Rhizopus spec. (WS 90027) were added. 5.0 ml of the solution thus obtained was mixed with 0.02 ml of serum in the one case and 0.02 ml of a cholesterol standard solution containing 200 mg% cholesterol in the other.
• the cholesterol content of the serumcontaining specimen amounted to l54 mg% total cholesterol.
• the control determination performed with cholesterol esterase from Candida rugosa ATCC 14830 instead of cholesterol esterase from Rhizopus spec. (WS 90027) gave the same value.
• Method of determining total cholesterol or bound cholesterol in a sample comprises treating said sample with cholesterol esterase obtained from a micro-organism, thereby releasing the bound cholesterol, and then determining the resulting cholesterol content of said sample using a standard determinatiion.
• microorganism is Candida rugosa ATCC M830.
• micro organism is Rhizopus spec. WS 90027.
• microorganism is Aspergillus spec. WS 90030.
• microorganism is Acrirmmyces aureovern'cillium WS 90002 Aclinamyces cyaneofuscatus WS 90003 Aclinamyces griseomycini WS 90004 Actinomyces longispurus-fl.
• WS 90005 Aclinomyces malachr'rr'cu:
• WS 90006 Acn'namyces mseolus WS 90007 Acn'nomyces mxym'cr'ni WS 90008 Acn'nomyces variabilis WS 90009 Slreplomyces spec.
• WS 900l0 Srrepmmytes aulotrophicus WS 900] l Srrepmmyces canescens WS 900 l 2 Srrepmmyces charrreusl's IS 90013 Strepromyces michiganensl's WS 900M Streptomyces murinus WS 900
• cholesterol oxidase is from Nocardia erythropolis ATCC 17895, Nocardia eryth ropolis ATCC 4277, Nocardia formica l48l l or Proactinomyces erythropolis NClB 11.
• said microorganism has been cultivated on a cholesterol ester containing nutrient medium.
• Reagent composition for the determination of cholesterol in a sample comprises cholesterol esterase obtained from a microorganism and a system for the determination of free cholesterol.
• Reagent composition as claimed in claim 13 wherein said microorganism is Candida rugoas ATCC 14830, Rhizopus spec. WS 90027 or Aspergillus spec. WS 90030.
• Reagent composition as claimed in claim 12 comprising cholesterol esterase,
• Reagent composition as claimed in .claim 14 containing peroxidase, a chromogen, a buffer,
• Reagent composition as claimed in claim 12 wherein said composition comprises groups, to result in the formation of hydrazone.

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