GB1573918A - Method of detecting the presence of protein in blood samples - Google Patents

Description

PATENT SPECIFICATION

( 21) Application No 23098/79 ( 22) Filed 26 Oct 1976 ( 62) Divided out of No 1573915 ( 31) Convention Application No 727633 ( 32) Filed 29 Sept 1976 in ( 33) United States of America (US) ( 44) Complete Specification published 28 Aug 1980 ( 51) INT CL 3 GOIN 33/50 ( 52) Index at acceptance GIB BA ( 54) A METHOD OF DETECTING THE PRESENCE OF B-PROTEIN IN BLOOD SAMPLES ( 71) We, RESEARCH CORPORATION, a Corporation organized under the laws of the State of New York, United States of America, of 405 Lexington Avenue, New York, New York 10017, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the

following statement:-

This invention relates to a method of discriminating between blood samples.

It is an object of this invention to provide a method for detection of a specific protein referred to herein as B-protein.

Detection of B-protein is accomplished by addition to the serum of a reagent containing a radioactively tagged form of a low molecular weight binding protein This binding protein binds to any B-protein present in the serum sample, and the Bprotein/bound protein complex can be detected by radioactivity counting techniques The low molecular weight binding protein is produced during interaction of an extract of Co A-SPC from Bakers' yeast with substrates therefor.

Reference is directed to our copending Application No 44355/76 Serial No.

1573915 which describes the preparation of the said low molecular weight binding protein The use of this low molecular weight binding protein is a method of detecting cancer is described in our copending Application No 7923096 Serial No 1573916 A specific type of protein normally present in human serum has been observed to interact with a low molecular weight protein component (referred to herein as the binding protein) released from the coenzyme A-synthesizing protein complex (Co A-SPC) of Bakers' yeast In certain patients the serum protein appears to be modified, or a protein with somewhat similar characteristics is produced and released into the blood, which also interacts with the low molecular weight protein of yeast This protein found in the serum of certain individuals has tentatively been designated the Bucovaz-protein (B-protein).

The normal protein and the B-protein have similar properties Both interact with the binding protein mentioned above.

Moreover, the B-protein and the specific protein present in the serum of all individuals migrate in an electrical field in the same general area as the y-globulin fraction of human serum Both proteins have a molecular weight of 140,000 to 160,000 and have the same pattern of separation in a 3 % to 10 % continuous sucrose gradient Also both proteins are precipitated by addition to the serum of ammonium sulphate to 50 % of saturation.

Furthermore, as the titer of B-protein in the serum increases, the amount of its normal counterpart appears to decrease in a proportional manner Although present information is not adequate to make a definite statement, and without intending to limit this invention in any way, it is speculated that the B-protein represents a modification of the yet unidentified protein of normal serum which interacts with the binding protein produced from Co A-SPC.

However, although the B-protein is similar to the normal serum protein, differences in the properties between the two are sufficient for separation and identification of the B-protein.

The specific low molecular weight binding protein described above is useful in that it can be used in radioactively tagged or labelled form in a method of discriminating between blood samples which comprises detecting the presence of B-protein and discriminating against normal protein by the addition to a patient's serum sample of a reagent containing the said specific low molecular weight binding protein in a radioactively tagged form This binding protein is preferably contained in a reagent comprising Co A-SPC The released binding protein binds with both the normal protein and B-protein in the serum.

( 11) 1 573 918 1,573,918 Subsequent partial denaturation of the bound protein complexes serves to discriminate between the two types of proteins based upon solubility differences thereby induced.

According to the present invention there is provided a method of discriminating between blood samples of one predetermined type and blood samples of another predetermined type on the basis of their comprising B-protein content separating serum from each blood sample and assaying each serum sample for Bprotein (as herein described) using a standard assay technique.

The standard assay technique preferably comprises treating all the serum samples with radioactively tagged binding protein which binds with the protein in the serum samples, partially denaturing the binding protein/serum protein complex so as to discriminate between B-protein/binding protein complex and the normal protein/binding protein complex and sensing the level of radio-activity of the partially denatured binding protein/serum protein complex in each sample.

The partially denatured binding protein/serum protein complex is preferably separated from each sample before its level of radioactivity is sensed.

In one form of the invention the assay technique comprises treating all the samples with radioactively tagged binding protein which binds with the protein in the serum samples, partially denaturing the binding protein/serum protein complex so as to discriminate between the B-protein/binding protein complex and the normal protein/binding protein complex, comparing the radioactivity of the said partially denatured binding protein/serum protein complex from each sample with that of a sample of partially denatured binding protein/serum protein complex prepared in the same way from a known serum sample containing B-protein or one not containing B-protein.

The treatment with radioactively tagged binding protein preferably comprises adding to each serum sample a reagent comprising an extract of coenzyme A-synthesizing protein complex (referred to herein as Co ASPC) from Bakers' yeast which has been tested by the procedures of Tests 7 and 8 herein and shown to have release activity for binding protein, and amounts of radioactively tagged substrates for the said extract which interact with the said extract to produce radioactively tagged binding protein.

The said substrates are preferably adenosine triphosphate (referred to herein as ATP) or a salt thereof, D-pantothemic acid or a salt thereof, and L-cysteine or a salt thereof, and there may be used for example ( 35 S)-L-Cysteine or ( 14 C-U)-Lcysteine or ( 14 C)-D-pantothenic acid.

The p H of the said reagent is from 6 2 to 7.6 and the reagent preferably further comprises a buffer which maintains the said p H value.

The sensing of the level of radioactivity is preferably performed by radioactivity counting.

The method may thus comprise reacting the said reagent and serum sample, partially denaturing the binding protein/serum protein complex produced thereby, separating the denatured from the nondenatured complex, and measuring the amount of said complex which has been denatured.

The said partial denaturing may comprise heating, or addition of a denaturing agent or a combination thereof.

A preferred form of the method comprises mixing from 0 1 to 5 ml of the reagent per 0 01 to 0 3 ml of the blood serum sample, incubating the mixture at a temperature of from 0 to 400 C for from 1 5 to 3 5 hours, terminating the incubation reaction by heating the incubation mixture for from I to 10 minutes at a temperature of from 65 to 100 "C, centrifuging the mixture, adding to the supernatant liquid from 0 1 to ml of a 1 to 50 % by weight aqueous solution of trichloroacetic acid per said volume of blood serum, and measuring the radioactivity of that portion of the protein complex which has been denatured.

The mixture of trichloracetic acid and the supernatant liquid is preferably heated for from 1 to 10 minutes at a temperature of from 65 to 1000 C.

The method preferably comprises, after the said partial denaturing, filtering the complex-containing solution, and measuring the radioactivity of the said complex which remains on the filter.

The reagent is preferably prepared by a method which comprises freezing and subsequently thawing Bakers' yeast and subjecting the said thawed yeast to agitation to release Co A-SPC from the said yeast.

Thus the method may comprise freezing and subsequently thawing Bakers' yeast, subjecting the said thawed yeast to a first agitation, sufficient substantially only to release endogenous proteins and not any substantial amounts of Co A-SPC, removing the liquid phase from the solid phase, resuspending the said solid phase, and subjecting the said resuspended solid to a second agitation to release Co A-SPC.

Preferably between the thawing and the agitation a salt is present, the amount and nature of the salt being such as to ensure that Co A-SPC is recoverable from the thawed yeast.

3 17 91 3 After the agitation or the second agitation the yeast solution is preferably centrifuged, for example at 6000 or 8000 g and the supernatant liquid containing the Co A-SPC is collected.

The agitation may comprise mechanical stirring at a temperature of from 0 to 120 C.

The first agitation is preferably carried out for from I to 4 hours.

In a preferred form of the invention the reagent comprises from 0 04 to 0 06 ml of the extract of Co A-SPC from Bakers' yeast, 1.5 to 5 m M of ATP or a salt thereof e n the disodium salt, 0 5 to 0 6 m M of Dpantothenic acid or a salt thereof, e g, the hemicalcium salt, 0 05 to 0 15 m M of Lcysteine or a salt thereof, and up to 0 8 ml of a buffer which maintains the p H of the reagent in the range of from 6 5 to 7 2 each quantity being per 1 ml of the total reagent, the balance being distilled water, the Lcysteine being in the ( 35 S), or ( 14 C-U-)radioactive form or the D-pantothenic acid being in the ( 14 C)-radioactive form.

The coenzyme A-synthesizing protein complex utilises L-cysteine, D-pantothenic acid and ATP as substrates The following explanation of the sequence of reactions leading to release of the aforementioned binding protein is of a theoretical nature only and is not meant to limit this invention in any way The initial reaction catalyzed by the Co A-SPC is thought to be between the IBphosphorous group of ATP and the 4 ' hydroxyl group of pantothenic acid resulting in the formation of Co A-SPC bound ADP-4-pantothenic acid The aamino group of cysteine is then thought to react with the carboxyl group of the pantothenic acid moiety At the time of reaction, cysteine is thought to be decarboxylated forming Co A-SPC bound dephospho-Co A Dephospho-Co A is thought to be either phosphorylated and released as Co A, or hydrolyzed to yield what appears to be 4 '-phosphopantetheine bound to the low molecular weight protein component of the Co A-SPC, which then detaches from the complex The molecular weight of the binding protein is in the range of 8000 to 18,000, and most preferably 10,000 to 15,000 (weight average).

One function of the Co A-SPC apparently is the synthesis of Co A: the other may be associated with the synthesis of acyl-carrier protein The low molecular weight binding protein is suspected of being related to the latter of these two functions of Co A-SPC In regard to the B-protein assay, the low molecular weight protein is what is referred to as the binding protein.

The following preferred preparative techniques can be used to provide the Co ASPC for the assay of this invention These methods involve first freezing and subsequently thawing Bakers' yeast The thawed yeast is then subjected to agitation whereby Co A-SPC is progressively released from its cellular structural affinities The agitation can be provided by conventional techniques such as mechanical stirring or bubbling a gas, e g, air, CO 2, or N 2 through the mixture Many variations of this basic process are possible Other methods of breaking the yeast cell, such as grinding, the use of ultrasonics or pressure have proved to be unsuccessful.

The first specific procedure is as follows:

Bakers' yeast is crumbled into fine particles and frozen For example, freezing can be performed for from 1 to 6 hours, preferably from 4 to 6 hours, in diethyl ether ( 1.5-2 01)-CO, ( 5-7 Ibs dry ice) mixture.

The time and temperature of the freezing step are not critical) Commercially available Federal Brand and Fleischman yeast have been successfully utilised The frozen yeast is allowed to thaw under ambient conditions to room temperature If a freezing medium such as the CO 2/diethyl ether mentioned above is used, residual diethyl ether and CO 2 should be removed, e.g, by vacuum treatment to avoid denaturation of the protein The thawed yeast is then agitated, e g, by stirring to release Co A-SPC If desirable, a stabilising agent such as KCI or a similarly effective salt may be added Preferably up to 15 g of KCG, more preferably from 1 to 10 g, per pound of Bakers' yeast are added to the thawed homogenate Typical Bakers' yeast samples range from 1 to 4 pounds, preferably from 2 to 3 pounds, but the sample size, of course is not critical The conditions used during the agitation step depend upon the rate of agitation and are chosen to effectively release the Co A-SPC without attendant denaturation of the enzyme and other proteins in the homogenate Preferred conditions comprise stirring for from 6 to 25 hours; more preferably from 15 to 20 hours at a temperature of from 0 to 120 C, more preferably 0 to 3 C After stirring, the Co A-SPC-containing liquid may be removed from the solid components by centrifuging for from 10 to 30 minutes at 6,000 to 8,000 g Subsequent conventional purification procedures can then be employed on the supernatant liquid For instance, the Co A-SPC-containing liquid can be decanted through several (e g, 3-6) folds of cheesecloth Using such a procedure, about 350-375 ml of crude Co A-SPC extract has typically been obtained from a three pound sample of Bakers' yeast The crude extract has been stored for one year at cryogenic temperatures of from approximately -90 to I 1,573,918 1.573918 -100 C without losing its capacity of Co A and for release of the binding protein.

The second procedure which is a modification of that described above, is a preferred method It is similar to the method described above but in addition provides for the further purification of Co A-SPC This method is as follows:

Bakers' yeast is crumbled, frozen and thawed as above However, prior to the foregoing agitation step, the thawed yeast is subjected to a preagitation, wherein the aforementioned stabilizers, such as KCI, may also be incorporated This first agitation is provided to remove endogenous substrates from the yeast into the liquid phase, and not to effect release of significant amounts of Co A-SPC Thus, it is generally appplied for a much shorter period of time For example, stirring for from I to 4 hours at temperatures such as 0 to 120 C can be employed The liquid phase is then removed, for example, by one or more centrifugations The solid phase is then resuspended in aqueous solution For example, Buffer A, defined hereinbelow, may be used The resultant mixture is then agitated to release the Co A-SPC and subsequently treated as in the first method.

This second agitation can be for a shorter period of time than used for the sole agitation in the first method For example, stirring for from 11 to 13 hours can be used.

The purified extract has been found to represent a 973-fold purification of the Co A-SPC.

One embodiment of the reagent to be used in this invention comprises the Co ASPC extract and substrates thereof which interact with the extract to produce the binding protein Such substrates include ATP or a salt thereof, such as the disodium salt, D-pantothenic acid or a salt thereof, such as the hemi-calcium salt, and Lcysteine or a salt thereof These four components are preferably used together to react to produce the binding protein To speed up the reaction, the mixture may be incubated The ratio of amounts of the components and the p H of the reagent are not critical as long as the binding protein is produced.

A preferred p H range is from 6 2 to 7 6 more preferably from 6 5 to 7 2 One specific formulation found to produce satisfactory results in the assay of this invention is as follows: from 0 01 to 0 1 ml, preferably 0 04 to 0 06 ml, of Co A-SPC extract; from 0 01 to 10 m M, preferably from 1 5 to 5 m M of ATP or a salt thereof; from 0 01 to 1 0 m M, preferably from 0 5 to 0.6 m M, of D-pantothenic acid or a salt thereof; and from 0 01 to 0 6 m M, preferably from 0 05 to 0 15 m M of Lcysteine or salt thereof, per I m L of reagent, the remainder being water.

Control of the p H where required may be accomplished by addition of a conventional buffer The nature of the buffer is also not critical as long as the production of the binding protein is not prevented and the appropriate p H range is maintained.

Preferred conventional buffers include a solution of from 0 001 to 250 m M Trisacetate; from 0 01 to 50 m M magnesium acetate, and from 0 001 to 125 m M K Cl (this mixture being referred to herein as Buffer A); or a solution of from 0 001-250 m M KH 2 PO 4 and from 0 01 to 50 m M magnesium acetate For the specific formulation of the reagent mentioned above, up to 0 8 ml, preferably from 0 3 to 0.5 ml of such buffers can be added per I ml of total reagent solution.

It is also preferred that all solutions be formed using distilled and/or deionised water Moreover, since ATP is so acidic it can be provided in the reagent from a stock solution having a p H closer to that desired for the final reagent, such as 7 1 to 7 3 e g, around 7 2 Adjustment can be achieved by addition of a compatible base such as KOH, or Na OH.

One of the three substrates should be tagged in some manner so that the binding protein in the reagent is readily detectable.

Preferably radioactive tagging is used.

Suitable tagging can be achieved by incorporation of ( 35 S)-, or ('4 C-U)-L cysteine, ( 14 C)-pantothenic acid as the substrate in the reagent ATP can also be tagged using its -phosphorous group.

For the assay, a serum sample and the reagent are first mixed together, the order of mixing being non-critical The relative amounts of the reagent and serum are not critical but should be chosen so that the serum protein/binding protein complex is produced For example, for the specific formulation described above, from 0 1 to 5 0 ml, preferably from 0 5 to 2 0 ml, of reagent per 0.01 to 0 30, preferably 0 04 to 0 06 ml of serum sample is preferred but other proportions are also usable Of course, the ratio of the amount of reagent to the amount of serum sample will vary with the sensitivity of the radioactivity measurement, with the manner of performing the assay itself, e g, automated vs manual; with the specific composition of the reagent; with the specific technique used in the assay itself, e g, the particular filtration methods used; and other similar considerations.

After the mixing of the reagent and serum sample, the mixture is allowed to react to produce a sufficient amount of serum protein binding protein complex for subsequent measurement in the assay The 1,573,918 reaction can be conventionally aided, for example, by incubation The incubation conditions are chosen to enhance the appropriate reaction to form the complex without denaturing the active ingredients A large variety of time and temperature combinations are of course possible The lower the temperature the longer the incubation must be to produce amounts of binding protein complex equal to that produced at higher temperatures, and vice versa The choice of the incubation conditions is not critical and is determined by considerations of convenience, expense, efficiency, time and detection sensitivity.

Thus, quite low temperatures can be used if the attendant long incubation times are acceptable Preferred incubation conditions include the use of temperatures of from 0 to 400 C more preferably 30-370 C for from 1.5 to 3 5 hours.

Once the serum protein/binding protein complex has been produced, partial denaturation of the complex is used to discriminate between normal serum protein/binding protein complex and Bprotein/binding protein complex This denaturation differentially alters these two complexes so that the B-protein binding protein complex will be denatured significantly more or less than is its normal serum protein binding protein counterpart, generally more Which complex is denatured to the greater degree depends somewhat on the denaturation technique used In general, any conventional denaturation technique should be applicable For example, a heating treatment may be used and/or a chemical denaturation agent, such as trichloroacetic acid (TCA) and other similar conventional agents, can be added to the protein complex-containing solution Of course, the complexes should only be partially denatured so that a significant difference in the amounts of denatured complexes (Bprotein/binding protein vs normal protein/binding protein) exists permitting detection of the presence of the B-protein.

This difference in solubility, i e, denaturation properties, could be due to subtle differences in configuration and charge of the B-protein.

A preferred denaturation procedure in accordance with the above requirements comprises heating the aforementioned incubated reagent/serum sample mixture to a temperature such as from 65 to 100 C, preferably 68 to 70 'C for from 1 to 10 minutes preferably 3 to 6 minutes.

Employment of such high temperatures also serves to terminate the incubation reaction.

After cooling the solution e g, to less than 300 C measurement of the amount of denatured protein can be performed at this time, but because of the large amount of endogenous and other non-protein-complex protein which is denatured initially it is preferred to first remove the denature protein at this time e g, by filtration or centrifugation (e g, at 2,000 to 2,300 rpm for from 4 to 6 min) Most of the yeast protein is thus removed as a precipitate The supernatant liquid or filtrate contains the radioactively labelled binding protein/normal protein complex and the radioactively labelled binding protein/Bprotein complex, or just the former, depending on the source of the serum.

Thereafter, effective discrimination between the two complexes can be performed by additional partial denaturation, e g, by addition of a conventional denaturation reagent such as TCA or alcohols For example, for the specific formulation described above, from 0.1 to 5 ml, preferably 1 to 2 ml, of from 1 to % by weight aqueous solution of denaturation agent (e g, TCA) per ml of supernatant liquid or filtrate can be added, preferably with agitation.

At this stage the two complexes will be denatured to different extents The final step of the assay involves measurement of the total amount of complex which is denatured Comparison with the results of similar assays on normal and B-protein containing control serum samples, as described below, enables discrimination of the samples into those containing B-protein and those not containing it First, of course, the resulting protein precipitate (denatured protein complexes) is separated in a conventional manner from its still solubilised, undenatured counterpart, for example, by centrifugation or preferably by filtration Suitable conventional filters include Whatman (Registered Trade Mark) 3 MM filter paper discs or Millipore (Registered Trade Mark) Conventional washing (e g, 3 to 5 washes using 2 to 3 ml of water) and drying (e g, at 80 to 90 'C for from 10 to 20 minutes) of the precipitate should precede the denatured protein measurement Of course, scorching must be avoided The dried discs are then analysed for the presence of denatured binding protein/serum protein complex in conventional fashion.

For example, when the binding protein is radioactively tagged, the dried filters can be transferred to scintillation liquid vials, and the radioactivity level measured by scintillation counting Comparison with control values of radioactivity levels allows ascertainment of the presence of B-protein.

For each set of unknown serum samples tested, predetermined equivalent normal serum and B-protein containing samples are run under identical conditions as controls.

s 1,573,918 Preferably for the specific formulation and denaturation procedure described above the normal serum range is from 200 to 500 cpm, for example The B-protein serum range is 900 to 1500 cmp, for example Sera which fall within the lower range are considered normal Those in the higher range are considered to contain B-protein.

Whenever counts are close to the range limits, assays should be re-run If no exact determination can be made, the serum should be considered "unknown".

However, generally the counts are always obviously located in either the high or low range and little difficulty is encountered in differentiating normal from B-protein samples Variations in the radioactivity of the tagged substrate do not affect the assay accuracy since any changes in the radioactivity level are reflected in both the normal and B-protein controls.

Using this procedure, considerably less radioactivity and protein are trapped on the filters when B-protein is absent than when it is present due to the differing effects of the specific denaturation treatments on normal and B-protein These effects differentially alter the proteins so that the binding protein/B-protein complex is adhered on the filter to a much greater degree than is the normal protein counterpart This is thought to be due to the fact that normal protein is more resistant to this denaturation by the heating/TCA treatments than is the B-protein Consequently, B-protein is the less soluble protein after the treatment so that a greater quantity of the normal protein passes through the filter while more of the B-protein is trapped on the filter Since approximately the same quantity of radioactively labelled binding protein interacts with the serum protein of all samples a higher level of radioactivity and protein is detectable on the filter when Bprotein is present in the serum.

The serum samples are recovered from blood which has been allowed to coagulate and centrifuged to separate the packed cells from the serum A typical procedure is described in Example 1 The serum should not be extensively hemolyzed, as indicated by a dark red colour, because the attendant red coloured species interferes with the assay by producing quenching of the radioactivity Lipemic serum, however, does not interfere.

As indicated above, many modifications of the above assay procedures can also be employed For example, after the incubation of the specifically formulated reaction mixture described above, the aforementioned appropriate amounts of denaturation agent may be added as a first denaturation treatment which also serves to terminate the reaction The mixture can then be subsequently subjected to denaturation by heating as described above, and subsequently be cooled The washing and filtering operations are then carried out.

As a result of this reversal of the order of denaturation treatments, the levels of radioactivity detectable are reversed Serum from patients with B-protein register a lower level of radioactivity on the filter than do those without B-protein Comparisons with control samples subjected to the same procedures in a manner analogous to that mentioned above, are carried out to perform the assay.

Another particularly preferred modification can also be used The reagent is incubated as above but without addition of the serum sample This procedure permits reaction of the Co A-SPC to form Co A and the low molecular weight binding protein The resultant reaction mixture is then passed through a conventional molecular weight cutoff filter or column. The weight average molecular weight cutoff

is arranged to be from 20,000 to 100,000 preferably 30,000 to 50,000, so that the low molecular weight binding protein is passed through The resultant filtrate is then used as a reagent for assay in precisely the same way as the reagent described above except that shorter reaction incubation periods at the same temperatures can be used.

It is also possible to successively fractionally filter this filtrate to isolate the preferred 8,000 to 18,000, more preferably 10,000 to 15,000, molecular weight fraction for the binding protein For example, this can be accomplished by passing this filtrate through a filter which passes only substances having molecular weights lower than 50,000 The resultant filtrate can then be passed through a filter passing only substances of molecular weights lower than 1,000 The resultant material collected on the filter is washed to purify the binding protein of interest.

The characteristics of the low molecular weight binding protein may be summarised as follows:

1 It interacts with B-protein of serum and its apparent counterpart in normal serum.

2 It is released from Co A-SPC during the course of reaction.

3 It has a molecular weight in the range of 8,000 to 18,000.

4 It has been shown to be a component of Bakers' yeast.

It is heat stable to temperatures of 700 C.

6 It has not been shown to bind with any protein other than B-protein under the conditions of the assay The following proteins were used to test this property:

albumin, steapsin, amniotic fluid proteins, spinal fluid proteins, human milk protein, 7 1,5 73,1 7 cow's milk protein, a-globulin (human), fibrin, peptone, histones (IIA, III, IV), hemaglobin, R Nase, pyruvate kinase, pepsin, casein, Jack bean meal protein, protein hydrolysate (commercial), urease, gramicidin, ribosomal protein and fibrinogen.

7 It contains 4 '-phosphopanthetheine within its structure.

The invention may be put into practice in various ways and a number of specific embodiments will be given to illustrate the invention by way of example only with reference to the accompanying drawings, in which:

Figure 1 shows the change in radioactivity with time of Co A-SPC bound radioactivity for two Co A-SPC preparations prepared in accordance with the invention, and Figure 2 shows the gel filtration pattern of Co A-SPC (- -) having release activity, and of Co A-SPC having bound radioactivity EXAMPLE I

This describes the preparation of Co ASPC from Bakers' yeast, the Co A-SPC being useful to make up a reagent for use in the method described below in Example 3.

The reagent is described in Example 2.

Three pounds of Bakers' yeast were crumbled and then frozen for four to six hours in a diethyl ether/CO 2 mixture The frozen yeast was allowed to thaw, and residual diethyl ether and CO 2 were removed by vacuum Fifteen grams of K Cl were added to the homogenate, and the mixture was stirred at 00 to 40 for 17 hours.

Following the stirring step, the homogenate was centrifuged for 20 min at 7,700 g, and the supernatant liquid layer was decanted through several folds of cheesecloth The volume of the crude extract varied between 350 and 375 ml, depending upon the water content of the yeast The crude extract was stored at cryogenic temperatures of -1000 C The Co A-SPC prepared as described retained its capacity to synthesize Co A and to release the binding protein for several months.

EXAMPLE 2

This describes how the Co A-SPC of Example I is used to make a reagent for use in the B-protein Assay procedure described in Example 3 The reaction mixture is one in which l 35 Sl-L-cysteine is used as the radioactive tracer (substrate) lM 4 C-U-l-Lcysteine or l 14 Cl-D-pantothenic acid can be used in place of l 35 SlL-cysteine.

A reaction mixture containing the following components was made up 2 m M disodium ATP, p H 7 2: 0 5 ml buffer A, p H 7.2 (containing 0 05 M Tris-acetate, p H 7 2:

0.01 M magnesium acetate; 0 02 M K Cl); 0 5 m M hemicalcium D-pantothenic acid; 0 02 m M l 3551-L-cysteine ( 60,000 cpm); 0 05 ml of the yeast extract containing Co A-SPC prepared in Example 1 and water to a total volume of 1 ml.

EXAMPLE 3

This describes the assay procedure.

Patient Population One thousand patients, both male and female, of all economic levels were included in the tests described in this example Most of patients were in the age group between and 70 Some patients, however, were as young as 13 and others were as old as 92.

Serum for Assay Blood from each patient was collected in ml red stoppered, silicone coated Vacutainer (Registered Trade Marks) tubes.

No anticoagulant was added The clot that formed was removed, and the blood was centrifuged Following centrifugation, a Seraclear plug (Technicon) was inserted to separate the packed cells from the serum.

Serum which was extensively hemolyzed was discarded and a new sample collected.

The red colour caused by hemolysis interferes with the assay by producing quenching of the radioactivity measurement Lipemic serum, however, does not appear to have a significant effect on the results of the assay Serum could be stored at -200 C for several weeks or stored for longer periods of time at a cryogenic temperature of -1000 C Normal and cancer controls were chosen by screening serum from a number of individuals with and without cancer These controls were used to provide the range of values for both normal and cancer samples.

To the reaction mixture of Example 2 was added 0 05 ml of the serum being tested, and the total mixture was incubated at 360 C for 2 hours The reaction was terminated by heating the tubes in an H 20-bath at 68 VC to 70 C for 5 min The tubes were then cooled to room temperature followed by centrifuging at approximately 2,200 rpm for min in a Model CL International Clinical Centrifuge This procedure removed most of the yeast protein as a precipitate The remaining supernatant liquid of the reaction mixtures in which normal serum was added contained the l( 35 S)l binding protein normal protein complex Whereas, the supernatant liquid of the reaction mixtures in which serum containing B-protein was added contained both l( 35 S)l-binding protein normal protein and l( 35 S)l-binding process B-protein Two milliliters of 10 %.

aqueous TCA solution were added to each tube containing the supernatant liquid and the tubes were shaken to assure proper mixing of the TCA The resulting protein I 1,573,918 1,573,918 precipates, which were primarily serum protein were recovered by filtration using a Millipore filtering apparatus and Whatman No, 3 MM paper discs The precipitates collected on the discs were washed 4 times with approximately 2 ml of water per wash.

The discs containing the serum protein precipitates were dried in an oven at < 1000 C Precautions were taken to prevent scorching the discs The dried discs were then transferred to scintillation vials containing a scintillation liquid described by Hoskinson and Khorana, Journal of Biological Chemistry No 240, pages 21292135, ( 1965), and the radioactivity levels were measured in a Nuclear Chicago liquid scintillation counter.

With each set of unknown serum samples tested, a set of predetermined normal serum and B-protein serum samples were run as controls The cancer serum samples produce a radioactively labelled denatured B-protein/binding protein complex which is used as a standard This standard forms the subject matter of our copending application No 7923097 Serial No 1573917, to which reference is directed The normal serum range was 200 to 500 cpm The Bprotein serum range was 900 to 1500 cmp.

Sera which fell within the lower range was considered normal Those in the higher range were considered to contain B-protein.

Whenever counts were close to the range limits, the assay was re-run If no exact determination could be made, the serum was labelled "unknown" However, generally the counts were obviously located in the high or low range and there was little difficulty in identifying normal from Bprotein samples.

EXAMPLE 4

This is similar to Example 1 but describes a modified procedure for the preparation of Co A-SPC from Bakers' yeast Three pounds of Bakers' yeast were crumbled and frozen for 4 to 6 hours in diethyl ether/CO, mixture The frozen yeast was allowed to thaw, and residual diethyl ether and CO 2 were removed by vacuum Fifteen grams of K Cl were added to the homogenate, and the mixture was stirred at 00 to 40 C for 3 hours.

This initial stirring procedure removed endogenous substrates from the Co A-SPC.

ml of the homogenate were transferred to another flask and stirred for an additional 12 hours This portion of the homogenate was used only as a basis to determine the degree of purification of the Co A-SPC The remainder of the homogenate was centrifuged at 7,700 for 20 minutes and the supernatant liquid, discarded A volume of 1:10 dilution of buffer A (Described in Example 2) equal to that of the discarded supernatant liquid was added to the pellets in the centrifuge tubes The pellets were resuspended, centrifuged and the supernatant liquid discarded The addition of buffer A was repeated, and the pellets were resuspended and pooled Stirring of the resuspended pellet material was continued for an additional 12 hours.

During this stirring procedure, Co A-SPC was progressively released from its cellular structural affinities.

EXAMPLE 5

Example 2 was repeated Co A-SPC of Example 4 Example 1.

but using the rather than EXAMPLE 6

This was the same as Example 3 except that the reaction mixture used was that of Example 5 rather than Example 2.

The following test procedures were used to both characterise the nature of the assay and to assure its effectiveness and validity.

Test 1 Assay for Co A-SPC Release Activity The composition of the reaction mixture used to assay for the Co A-SPC binding protein release activity was the same as described under Example 3 except serum was omitted from the mixture i e, the reaction mixture was as described in Example 2 20 ml of the reaction mixture were incubated at 360 C Samples were removed for assay at 30 minute intervals during the course of a 150 minute incubation period Termination of the reaction of each sample removed for assay was accomplished by the addition of 2 ml of %/ aqueous TCA solution and heating the samples at 950 C for 5 minutes The tubes were then cooled in an ice bath, and the precipitates were recovered by filtration using a Millipore filtering apparatus and Whatman No 3 MM paper discs The precipitates collected on the discs were washed 4 times with approximately 2 ml of water per wash The discs were dried in an oven at 1000 C and then transferred to scintillation vials for radioactivity determination.

Following the 150 minute incubation period, another sample was removed from the reaction flask and applied to a column of Sephadex G-200 Registered Trade Mark and eluted with buffer A at a flow rate of 1 ml per minute.

Test 2 Determination of Protein Protein concentration was determined by the method of Lowry et al, J Biol, Chem, 193, 265-275 ( 1951).

1,573,918 Test 3 Gel Filtration Approximately 40 g of Sephadex G-200, coarse (Pharmacia), were equilibrated for 4 days in buffer A A column ( 2 5 x 60 cm) was prepared from the material and allowed to settle for 2 days The column was kept at 0 to 40 C The Co A-SPC was eluted using buffer A at a flow rate of 1 ml per minute.

Test 4 Support for Protein-Protein Interaction One hundred ml of a reaction mixture with the same concentration of components as described under Test 1 were incubated for 2 hours at 360 C Following incubation, the reaction mixture was divided into two equal parts Half of the mixture was filtered using Amicon Centriflo cones with a cut-off at a molecular weight of 50,000.

Each filtrate was tested individually in the procedure of Example 3 in the B-protein by adding one ml of the filtrate to each of a series of tubes containing serum to be assayed The mixtures were then incubated at 361 C for 30 min Following the incubation, the reaction was terminated by heating the tubes at 680 C to 701 C for 5 min.

and the remainder of the procedure described in Example 3 was followed.

For the Co A-SPC to be functional in the B-protein assay, experimental evidence supports for contention that the Co A-SPC must have release activity for the low molecular weight protein (binding protein).

Evidence for protein-protein interaction between the binding protein and either the B-protein or its normal counterpart is highly suggestive, but not conclusive However, all evidence accumulated thus far would support a protein-protein interaction Of course, this theory is not meant to limit this invention in any way.

Test 4 2 A further sample of each filtrate was added individually to a second series of tubes containing serum to be assayed Also added individually to these reaction mixtures was 0 20 m M L-cysteine, Dpantothenic acid, ATP, Co A, dephosphoCo A, pantetheine and 4 'phosphopantetheine as possible inhibitors of the reaction.

An approximately 5-fold excess of each of the L-cysteine D-pantothenic acid, ATP, Co A dephospho-Co A, pantetheine and 4 'phosphopantetheine, when added to the reaction mixture, did not interfere with the B-protein assay This would be expected if the B-protein assay is a protein-protein interaction between the B-protein of serum and the radioactively labelled binding protein If some other radioactively labelled 4 '-phosphopantetheine were being transferred from the yeast binding protein to the B-protein, it might be expected that the unlabelled components added to the reaction mixture would interfere with the Bprotein assay.

Test 4 3 Additional evidence in support of the binding protein being the functional product of Co A-SPC in the B-protein assay was provided by filtering the other one-half of the reaction mixture through an Amicon ultra-filtration membrane UM-2 having a cut-off at a molecular weight of 1000 This filtration permitted the passage of only low molecular weight components of the reaction mixture into the filtrate In this case the filtrate, which did not contain radioactively labelled binding protein, was not functional in the B-protein assay.

Test 5 Molecular Weight Determination The Sephadex G-200 column prepared as described above under Test 3 was used The total bed volume, inner gel volume, volume of gel, and void volume of the column were determined A calibration curve (Andrew, Biochem J, 91 222-223 ( 1964), was used to estimate the molecular weight of the complex, the binding protein, B-protein and the specific protein of normal serum which interacts with the binding protein The calibration curve was obtained by chromatographing a mixture of glutathione (Sigma Chemical), bovine serum albumin (Sigma Chemical), bovine pancreas acchymotrypsin (Sigma Chemical), and bovine ribonuclease (Sigma Chemical).

Test 6 Inhibition of Co A-SPC Activity Inhibition of Co A-SPC activity was determined as described under Test 1 except the incubation time was 1 hour, and 0.20 m M Co A, dephospho-Co A, pantetheine and 4 '-phosphopantetheine were added individually to reaction mixtures.

Results Not all preparations of Co A-SPC release binding protein which is needed for the function of the B-protein assay Thus, each new preparation of Co A-SPC was tested in the following ways to determine if binding protein release activity was present.

Test 7 Profile of Co A-SPC with Functional release Activity The change in radioactivity with time of Co A-SPC bound radioactivity is shown in Figure 1 For this study, ( 35 S)-L-cysteine was used as the radioactive substrate The 1,573,918 experimental procedure was the same as described under Test 1 As shown in Figure 1, a 5 to 6 minute delay was observed before a measurable amount of protein-bound radioactivity, representing dephospho-Co A formation, was detected This lag period was followed by a linear increase, which in turn was followed by a levelling-off period.

In the majority of Co A-SPC preparations in which release activity was functional, a significant decrease in the level of bound radioactivity was observed during the second hour of incubation (broken line, Figure 1) With preparations of Co A-SPC which have lost release activity, the plateau region of the curve was maintained throughout the second hour of incubation (solid line, Figure 1) These two patterns were also seen if ('4 C)-D-pantothenic acid ( 14 C)-ATP was used in place of ( 355)-Lcysteine as the radioactive substrate in the experiment.

Following the 150 minute incubation period, another sample of the reaction mixture was removed from the reaction flask and applied to a column of Sephadex G-200.

Test 8 Gel Filtration The Sephadex G-200 elution pattern of a reaction mixture in which Co A-SPC with release activity was used is shown in Figure 2 Column fractions collected were assayed for protein by the method of Lowry et al.

(See Test 2) and for radioactivity As shown, two peaks of radioactivity were eluted from the column One peak containing the major portion of the protein and radioactivity was eluted in fractions 33 through 100 The second fraction of radioactivity was bound to a protein of lower molecular weight (fractions 106 to 160).

Utilising the method described by Andrew, (see Test 5) the higher molecular weight component which represented the Co ASPC had an estimated molecular weight in excess of 200,000 and the lower molecular weight protein (binding protein) had an estimated molecular weight of 10,000 to 15,000 A similar elution pattern to the one shown in Figure 2 was obtained whenever ( 14 C)-D-pantothenic acid or ( 35 S)-L-cysteine was the radioactive substrate in the experiment Co A-SPC preparations which did not show the decrease in radioactivity during the second hour of incubation, Figure 1, also did not show the presence of binding protein released from Co A-SPC (Figure 2) Only Co A-SPC preparations which exhibited release activity for the binding protein were functional in the Bprotein assay.

Test 9 Effect of Reaction Products and Related Compounds on Co A-SPC Activity Reaction products and compounds with related structures were tested as inhibitors of Co A-SPC bound dephospho-Co A formation as described under Test 6 Co A, dephospho-Co A, pantetheine and 4 'phosphopantetheine at a concentration of 0.20 m M inhibited Co A-SPC activity at least 9 Q 0/ Test 10 Effect of Reaction Components and Products on B-protein Assay The procedure described under Test 4 1 was followed The radioactively labelled binding protein and other low molecular weight components were collected in the filtrate obtained by filtering a reaction mixture using Amicon Centriflo cones with a cut-off at a molecular weight of 50,000.

Another filtrate was obtained which did not contain the binding protein, but contained reaction components of molecular weight of 1,000 or less by filtering a reaction mixture using an Amicon ultrafiltration membrane UM-2 as described in Test 4 3 Only the filtrate containing a radioactively labelled binding protein, when added to a series of tubes with serum to be assayed, provided a functional B-protein assay.

The addition of 0 20 m M L-cysteine, Dpantothenic acid, ATP, Co A, dephosphoCo A, pantetheine and 4 '-pantetheine to assay mixtures which contained the filtrate that had radioactively labelled binding protein did not interfere with the B-protein assay.

Claims (1)

1. WHAT WE CLAIM IS:-

   1 A method of discriminating between blood samples of one predetermined type and blood samples of another predetermined type on the basis of their Bprotein content comprising described serum from each blood sample and assaying each serum sample for B-protein (as hereinbefore described) using a standard assay technique.

   2 A method as claimed in Claim 1 in which the standard assay technique comprises treating all the serum samples with radioactively tagged binding protein which binds with the protein in the serum samples, partially denaturing the binding protein/serum protein complex so as to discriminate between B-protein/binding protein complex and the normal protein/binding protein complex and sensing the level of radioactivity of the partially denatured binding protein/serum protein complex in each sample.

   1.573,918 3 A method as claimed in Claim 2 in which the partially denatured binding protein/serum protein complex is separated from each sample before its level of radioactivity is sensed.

   4 A method as claimed in any one of Claims 1-3 in which the assay technique comprises treating all the samples with radioactively tagged binding protein which binds with the protein in the serum samples, partially denaturing the binding protein/serum protein complex so as to discriminate between the B-protein/binding protein complex and the normal protein binding protein complex, comparing the radioactivity of the said partially denatured binding protein/serum protein complex from each sample with that of a sample of partially denatured binding protein/serum protein complex prepared in the same way from a known serum sample containing Bprotein or one not containing B-protein or with two such samples using the same batch of radioactively tagged binding protein.

   5 A method as claimed in any one of Claims 2-4 in which the treatment with radioactively tagged binding protein comprises adding to each serum sample a reagent comprising an extract of coenzyme A-synthesizing protein complex (referred to herein as Co A-SPC) from Bakers' yeast which has been tested by the procedures of Tests 7 and 8 herein and shown to have release activity for binding protein, and amounts of radioactively tagged substrates for the said extract which interact with the said extract to produce radioactively tagged binding protein.

   6 A method as claimed in Claim 5, in which the said substrate are adenosine triphosphate (referred to herein as ATP) or a salt thereof, D-pantothenic acid or a salt thereof, and L-cysteine or a salt thereof.

   7 A method as claimed in Claim 5 or Claim 6 in which the radioactively labelled substrate is ( 35 S)-L-Cysteine or ( 14 C-U)-Lcysteine or ('4 C)-D-pantothenic acid.

   8 A method as claimed in Claim 5, 6 or 7 in which the p H of the said reagent is from 6 2 to 7 6.

   9 A method as claimed in Claim 8 in which the said reagent further comprises a buffer which maintains the said p H value.

   A method as claimed in any one of Claims 2 to 9 in which the sensing of the level of radioactivity is performed by radioactivity counting.

   11 A method as claimed in any one of Claims 5 to 10 which comprises reacting the said reagent and serum sample, partially denaturing the binding protein/serum protein complex produced thereby, separating the denatured from the nondenatured complex, and measuring the amount of the said complex which has been denatured.

   12 A method as claimed in any one of Claims 1 to 11 in which the said partial denaturing comprises heating, or addition of a denaturing agent or a combination thereof.

   13 A method as claimed in any one of Claims 5 to 12 which comprises: mixing from 0 1 to 5 ml of the reagent per 0 01 to 0.3 ml of the blood serum sample, incubating the mixture at a temperature of from 0 to 400 C for from 1 5 to 3 5 hours, terminating the incubation reaction by heating the incubation mixture for from 1 to minutes at a temperature of from 65 to 1000 C, centrifuging the mixture, adding to the supernatant liquid from 0 1 to 5 ml of a 1 to 50 % by weight aqueous solution of trichloroacetic acid per said volume of blood serum, and measuring the radioactivity of that portion of the protein complex which has been denatured.

   14 A method as claimed in Claim 13 in which the mixture of trichloroacetic acid and the supernatant liquid is heated for from 1 to 10 minutes at a temperature of from 65 to 1000 C.

   A method as claimed in any one of Claims 2 to 14, which comprises, after the said partial denaturing, filtering the complex-containing solution, and measuring the radioactivity of the said complex which remains on the filter.

   16 A method as claimed in any one of Claims 3 to 15 in which the reagent is prepared by a method which comprises freezing and subsequently thawing Bakers' yeast and subjecting the said thawed yeast to agitation to release Co A-SPC from the said yeast.

   17 A method as claimed in any one of Claims 5 to 15 in which the reagent is prepared by a method which comprises freezing and subsequently thawing Bakers' yeast, subjecting the said thawed yeast to a first agitation, sufficient substantially only to release endogenous proteins and not any substantial amounts of Co A-SPC, removing the liquid phase from the solid phase, resuspending the said solid phase, and subjecting the said resuspended solid to a second agitation to release Co A-SPC.

   18 A method as claimed in Claim 16 or Claim 17 in which between the thawing and the agitation a salt is present, the amount and nature of the salt being such as to ensure that Co A-SPC is recoverable from the thawed yeast.

   19 A method as claimed in any one of Claims 16 to 18 in which after the agitation or the second agitation the yeast solution is centrifuged and the supernatant liquid containing the Co A-SPC is collected.

   A method as claimed in Claim 19 in lo 1 1 1 12 1,573,918 12 which the centrifuging is carried out at 6000 to 8000 g.

   21 A method as claimed in any one of Claims 16 to 20 in which the agitation comprises mechanical stirring at a temperature of from 0 to 12 C.

   22 A method as claimed in any one of Claims 17 to 21 in which the first agitation is carried out for from I to 4 hours.

   23 A method as claimed in any one of Claims 16 to 22 in which the reagent comprises from 0 04 to 0 06 ml of the extract of the Co A-SPC from Bakers' yeast, 1 5 to 5 m M of ATP or a salt thereof, 0 5 to 0 6 m M of D-pantothenic acid or a salt thereof, 0 05 to 0 15 m M of L-cysteine or a salt thereof, and up to 0 8 ml of a buffer which maintains the p H of the reagent in the range of from 6.5 to 7 2, each quantity being per I ml of the total reagent, the balance being distilled water, the L-cysteine being in the ( 355), or ( 14 C-U-) radioactive form or the Dpanthothenic acid being in the ( 14 C)radioactive form.

   24 A method as claimed in Claim 23 in which the ATP is present as the disodium salt, and D-pantothenic acid is present as the hemicalcium salt.

   A method as claimed in Claim 23 or Claim 24 in which the buffer comprises 0.001 to 250 m M of Trisacetate, 0 01 to 50 m M of magnesium acetate and 0 001 to 250 m M of KCI.

   26 A method as claimed in Claims 23, 24 or 25 in which the ATP salt is added from a stock solution thereof having a p H of 7 1 to 7.3.

   27 A method as claimed in Claim I substantially as specifically described herein with reference to Example 3 or Example 4.

   KILBURN & STRODE, Chartered Patent Agents, Agents for the Applicants.

   Printed for Her Majesty's Stationery Office, by the Courier Press, Leamington Spa 1980 Published by The Patent Office 25 Southampton Buildings, London WC 2 A l AY from which copies may be obtained.

   1,573,918