CA1040080A - Low-molecular products method and device - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
This invention relates to a method and device for producing low-molecular products, especially for determining the concentration of low-molecular biological substances by means of enzyme reaction. The invention is characterized by the fact that after the reaction is complete, the used enzyme is separated from the low-molecular substances of the reaction mixture by means of ultrafiltration, conducted into a circulation system, and used again for enzymatic production of low-molecular substances.

Description

o This invention relates to a method o~ and a device fox determining the concentrat~on o~ low-molecular weight biological substances by enzymatic react~on.
A chemical reaction catalyzed by specific enzymes is often used for determining the concentration of low-molecular weight biological substances. In this method, the test sub-stance is mixed with suitable buffers and adjuvants, and the re-action that makes the quantitative determination possible is produced by the catalytic effect of the added enzyme. The de-termination is usually done by measuring the optical absorption.
The enzymes necessary for this æe commercially available, but are relatively expensive. In the known processes, the reaction mixture is discarded after the measurement, so that the expen-sive enzymes are lost.
The present invention in one aspect provides a method of determining the conaentration of low-molecular weight biolo-gical substances by enzymatic reaction, wherein the low-molecu-lar weight reaction products obtained as a result of the enzy-matic reaction, together with the low-molecular weight biologi-cal substance are separated from the enzyme after the enzymatic reaction by ultrafiltration, and wherein the enzyme used therein which emerges as ~ concentrate from the ultrafilter separately from the reaction mixture, is recycled and used again for enzym-atic reaction with low-molecular weight biological substances, the concentration of the biological substances being determined by means of the measured concentration of the low-molecular weight reaction products.
The method according to the invention makes full use of the fact that all enzymes differ so greatly in molecular weight from the adjuvants and test materials that they can be separated from them by means of ultra-filtration. Thus the enzyme remains ~ - 2 -o ent~rely in the concentrate, wh~le the lo~molecular wei~ht com-ponents in the filtrate and concentrate are present in equal concentration. 5ince the amount of ~iltrate is significantly greater than the amount of concentrate, the ratio of enzymes to low-molecular weight substances in the concentrate is signifi-cantly greater than in the reaction mixture. On the basis of this fact, the re-use of the enzyme in the form of the concen-trate is made possible.
In a preferred embodiment of the present invention there is provided, in a method for determining the concentration of low-molecular biological substances by reaction with an enzyme including the steps of contacting a low-molecular biological sample with an enzyme, thereby producing low-molecular reaction products, quantitatively measuring the concentration ofthe low-molecular reaction products and discarding the unreacted sample and the low-molecular reaction products after measuring, the im-provement of recovering a substantial portion of the enzyme com-prising ~eparating the low-molecular reaction products and unre-acted sample from the enzyme by a first ultra-filtration into a ~o filtrate containing substantially all of the low-molecular reac-tion products and unreacted sample and a concentrate containing the enzyme and a trace quantity of the low-molecular reaction products and unreacted sample, diluting the concentrate and sub-jecting the concentrate to at least one additional ultra-filtra-tion, recovering the enzyme therefrom substantially devoid of low-molecular reaction products and unreacted sample, and re-using the thus separated enzyme in the continuous ~ncentration determinations.
In a further embodiment of the present invention there is provided an apparatus for quantitatively determining the con-centration of low-molecular biological substan~es by reaction ~ - 2a -4(3~t30 ~ith an enz~me and product~on of lo~olecula~ reaction products including:
a source of low-molecular biological sample;
a source of reactive enzyme;
pump means for delivering a predetermined quantity of sample from the sample source to a reaction area;
pump means for delivering a predetermined quantity of reactive enzyme from the enzyme source to a reac-tion area;
means for mixing together the delivered smaple and the delivered enzyme including therewith a reaction area:
pipe means communicating the reaction area to a first ultrafilter for separating a filtrate containing substantially all of the low molecular reaction products and unreacted sample from a concentrate containing the enz~me and a trace quantity of the same ahd low-molecular reaction products;
a second ultra-filter for further concentrating the . enzyme and removing trace portions of sample and low-molecular reaction products therefrom;
pipe means communicating the concentrate of the first ultra-filter to a second ultra-filter;
a source of enzyme diluent intermediate the first and second ultra-filters for delivery of enzyme diluent to the concentrate passing from the first ultra-filter to the second ultra-filter;
pipe means communicatecl from the outlet of the second ultra-filter for delivering theseparated, diluted enzy~me substantially devoid of sample to the mixing means;

~ .
~ - 2b -~ o~
pipe means ¢ommunicat~n~ the ~iltrate o~ the ~irst ultra-filter to an outlet ~or discarding the unreacted low~molecular sample and thelow~
molecular reaction products, and means for determining the concentration of the low~
molecular reaction products ~or evaluation of:-: the concentration o~ the low~molecular biological sample.

- 2c -g~4Q0~3~
After ultrafiltration, however, remains o~ the low-molecular components of the reaction mixture can still be found in the concentrate According to the invention, the remains of dissolved low-molecular components oi the reaction mi~ture left in the concentrate after reaction and ultrafiltration can be determined on the basis of the concentration measured after the reaction~ and they san be considered in the calculation of the next determination, using the enzyme le~t in the conoentrate. The amounts of low-molecular substances left in the concentrate can be determined from the determination of the concentration of low-molecular substances in a reaotion. This is done by seeing to it that in the next reaction~ which is per~ormed with the enzymcs left in the concentrate~ the amounts of low-mole-oular substances left in the concentrate are considered in the calculations, whereby the result of the determina-tion ln the second reaction performed with the same enzyme fraction can be corrected to such an extent, that the measurement is exact again. In this way, the determination of concentration can be performed with the re-used enzyme in exactly the same manner as with a fresh enzyme. The calculation can be performed simply in an electronic computer~ in which the data concerning the measurement performed previously with the same enzyme fraction are used~ However~ the oalculation of the fraction of low-molecular substances left in the concentrate presupposes that a number of such ~.Q4~
determinations will be performed in direct sequenoe.
In accordance with a speoial mode of operation of the invention, the proce~ can be perfor~ed in such a way that the concentrate flowing off the ooncentrate chamber of the ultrafilter can be-ultra-filtered again after dilution, whereby this ~tep oan be repeated ~everal times, and that the con¢entrate from the last ultraflltration i9 again used for pro-duoing low-molecular products by mean~ of enzyme reaction~
espeoially for enzymatic determination of low-moleoular biologioal substanoes. In the ~irst ultra-filtration~
the amount of low-molecular substances left in the con-oentrate is found from the ratio of concentrate to filtrate. I~ for example~ the proportion of concentrate to filtrate i9 1:10~ then only 1/10 of the low-molecular substanoes oontained in the reaotion mixture i9 left in the oonoentrate. If now the oonoentrate is diluted and ultrariltered again~ then it is found that in the case of the same proportion of oonoentrate to filtrate of 1:10 only 1/100 of the low-moleoular sub~tanoes contained in the reaotion mixture are present in the concentrate.
In many oases~ such a fraction of low-molesular sub-stances~ which affects the result~ oi the next determina-tion~ using the same enzyme~ can be taken into account.
The fraotion of 1/100 low-moleoular substances is in many cases already belo~ margin of error for the de-termination~ 90 that the enzyme can be re-used regard-less of the accompanying low-moleoular substances.
In any case, however~ this fraction is about equal again in the second and each subsequent instance of use of the enzyme~ so that consideration of an always constant correction factor in the measurement i~ sufficient.

However, if the concentrate i9 ultrafiltered again after dilution, then the smount of low-molecular substances in the concentrate is only 1/1000 oi the amount contained9 which i9 in nearly all cases lower than the margin of error for the determination.
Since in the case of repeated ultrafiltration, consideration of the fraction of low-molecular substan-ces left in the ooncentrate is not necessary~ it is also not neoessary in these ¢ases to take into account a result o~ the previous determination~ and thus~ in aocordance with the invention~ the concentrate of the la~t ultrafiltration can be stored before re-use.
This is especially advantageous if a di~oontinuable pro-oess i~ used.
Measurement of the reaction mixture can be performed in the same way a~ was used in already known methode~
when the reaotion mi~ture was thrown away after measure-ment. Aooording to the invention~ however~ the measure-me~t i9 preferably taken on the filtrate flowing off the flltrate ¢hamber of the fir~t ultrafilter. This is easily pos~ible, beoause the low-moleoular substanoes are present in the filtrate in the ~ame oonoentration as in the reaotion mi~ture. This also of~ers the advantage that the measurement is not fal~ified by the high-moleoular substanoes. The measurement i8 usually done with a photometer~ whioh produoe~ a heating of the material to be mea~ured. The faot that measurement is performed on the filtrate ofiers the advantage that the enzymes are not harmed by the heat effeot~ whioh is of great signifioanoe~ as these enzymes are to be re-used repeatedly.

~4~380 The method aooording to the invention is not limited to a determination of low-molecular biologi¢al ~ubstances by means of enzyme reaotion but oan al30 be used to produce low-molecular products by an enzyme reaction for other purposes.
In the device of the invention, for determining the ooncentration of low-molecular biological sub~tances by means of enzyme reaction, the enzyme i9 mi~ed with the sample in the u~ual way at a mixing point~ whereby the reaotion mi~ture is conveyed by means of pump-pressure through a reaction ~tretch, through a pipe, into a measur-ing point. In aocordance with the invention~ the pipe conducting the reaation mixture is connected to the entrance of the concentrate chamber of the ultraYilter~
whereby the exit of the ooncentrate ohamber is oonneoted with the mixing point, and a disoharge pipe i8 oonneoted with the filtrate ohamber. With suoh a devioe~ it is possible to perform a great number of oonoentration determinatlon~ in uninterrupted suooession~
whereby however~ i~ there is only one ultrafilter~ an eleotronio oomputing and storing devioe is u~eful~ for taking into aooount the fraotion of low-moleoular sub-stanoes lert in the oonoentrate and thus in the re-u~ed enzyme solution~ when viawing the previous determination perfor~ed with this enzyme fraotion. Aooording to a ~peoial mode of operation of the invention, therefore~
the apparatus i9 arranged in suoh a way that the outlet of the oonoentrate ohamber of the ultrafilter is connected with the inlet of the oonoentrate ohamber of a seoond ultrafilter, and a pipe for the solution agent i~
connected withtbe inlet of the conoentrate chamber of the seaond ultrafilter, and that the outlet of the ~4(~ 0 oonoentrate chamber of this seoond ultraiilter is oonne¢ted with the mixing point by means of a pipe~ ~nd that a disoharge pipe is oonneoted to the filtrate cham-ber of the seeond ultrafiltar. In thi~ case9 the fraction of low-moleoular ~ubstances leit in the conoentrate can generally be disregarded, 90 that an electronic oomputlng and storing deYice i~ not necessary. ~en~ in aooordance with the invention~ the pipe from the outlet Oir the oon-oentrate chamber of the second ultraiilter leads to the mixing point through the concentrate chambers oi other ultrafilters9 to the inlets of which pipes ior a solution agent are also connected~ then the aocuraoy of the determination is even iurther in¢reased.
~ he mea~uring point can be plaoed in iront of the inlet o~ the oonoentrate ohamber of the first ultra-filter; however~ aooording to the ~nvention the measuring point is plaoed within the disoharge pipe leading out of the iiltrate ohamber of the iirst ultra-~ilter~ whereby the advantages desoribed above are aohie~ed~ in that the enzyme to be re-used i9 protected against heating and thus grestly spared.
When several ultrafilters are used~ a storage oontainer for the enzyme oan be in~erted into the pipe leading from the concentrate ohamber of the last ultra-iilter to the mixing point. In this tank~ the enzyme or enzyme ~olution is stored before re-use.
In the drawing~ the invention is diagrammatioally explained~ with reierenoe to examples.
Fig. 1 shows a devioe for determining the oonoentration oi low-moleoular biologioal substanoe~ by means of enzyme reaotion~ using an ultrafilter. Fig. 2 ~hows this sort o~ devioa using several ultrafilter~.

~4(~U~O
In the oase of the dsvice represented in Fig. 1, there are three peristaltic pumps 1~ 2 and 3. Peristal-tic pump 1 draws in samples from the sample tank 4.
~he sample can be~ for example, a solution containing glucose. Peristaltic pump 2 draws in an adjuvant for the gluco~e to be determined~ e.g. a phosphate buffer eontaining o-anisidi~ as a chromogen~ from the ad~u~ant tank 5. Peristaltic pump draws in enzyme solution~
e.g. a solution o~ gluoose-oxydase and a peroxydase~
from the pipe 6~ which is connected with a ~torage tank 55 for fresh enzyme solution through a valve 53 and a pipe 54.
The solutions oonveyed by the peristaltio pumps~ operated~
for e~ample~ by synchronous motors~ are oonducted to a ~ixing point 8, and the mixture thus obtained is oonveyed through a pipe 7~ in whioh the reaotion takes plaoa. ~his pipe oan have~ for example~ an internal diameter of about 1 mm and be a polyethylene tube about 15 m long. ~he pipe with the reaotion mixture 7 leads into the inlet of the conoentrate chamber 21 of an ultrafilter 10 with a membrane 9. To the outlet of the oonoentrate ohamber 21 of the ultrafilter 10 there is attaohed a pipe 6~ through whioh the enzyme-rioh oonoentrate is oonduoted back to the mixing point 8 with the help of a peristaltio pump 3. At this time~
the valve 5~ is closed, whereafter all subsequent de-terminations are performed on the concentrate of the ultrafiltration, and taking into aocount the measure-ments of previous determinations. The low-molecular substanoes freed by the enzyme and those dye-containing ones formed upon the enzymatic reaction~ flow through a pipe 11 from the filtrate chamber 22 of the ultra-filter 10 to a photometer 12 (lamp 12~, flow cell 12"

and photo resistanoe 12"~ he signal given of~ by the photo resistor 12"t, whioh depend upon the ¢on-oentration of dye in the solution flowing through the flow oell 12"~ i9 registered in a logarithmic analog-digital-transformer and tran~formed into a digital ~ignal that i9 registered in a slide register 14, in the present oa~e a 16-stage slide register with 8 bits per stage.
The following should be considered in the analysi~
of the measurements given by the logarithmio analog-digital-transformer 13 and the measurement~ stored in digital form in the slide register 14.
In the oase of the above-indicated diameter o~
ths pipe 7, whioh i9 essentially identioal to the pipe 11 the pipe system has a volume of about 15 to 20 ml between the peristaltio pumps l, 2 and 3 on the one hand and the photometer 12 on the other~ so that it is pos~ible that a reaoted and there~ore oolored ~olution oould form in the photometer ~rom the suotioned samplep still during suotion of the substanoes ~rom the sample oontainer 4. If we assume that in the ultrafilter 10 : the reaotion mixturep in a volume ratio of l:9p is separated into an enzyme oonoentrate flowing away through the pipe 6 and an ultrafiltrate flowing toward the photometer and oontaining the oolored reaotion pro-duot~ and that the volume of the pipe 6 is 90 great that the enzyme solution used in the first test is re-used for the fourth testp then care must be taken that the value~ stored in the slide register 14 be stepped up by a timing generator 15 90 ~astp that the measurement entered into the slide regi3ter 14 for the first te~t should appear at the e~it of thi~ ~lide u~o register 14 at the moment ~hen the measurement for the fourth test i9 determinsd by the photometer 12.
Sinoe in the enzyme concentrate leaving the conoentrate ¢hamber 21 of the ultrafilter 10 and in the reacted solution leaving the filtrate chamber 22 of the ultra-filter 10 thro~gh the pipe 11 there i9 the same con-centration o-f colored reaction product~ the measurement appearing at the e~it of the slide register 14 for the ~irst te~t~ with a volume ratio of 1:9~ ~hould be reduoed, and the differenoe between the measurement for the fourth test and the reduced value ~or the first test should be indicated. Toward thiY end~ the digital signal that appears as an initial value in the logarith-mio analog-digital-transformer 13 for the measurement of the fourth test~ i8 transformed into an anulog signal in a digital-analog-transformer 16~ and the digital signal appeari~g simultaneously at the exit of the slide register 1l~ ~or the measurement of the first test is transformed into an analog signal in a digital-analog-transformer 17. This last analog signal i~ reduced in a reducer 18 acoording to the oited separation ratio of 1s9~ whereupon the difierence i9 obtained ~rom the analog signal giv.en by the digital-analog-transformer 16 and the signal given by the digital-analog-tran~former 17 and reduoed by the reducer 18. The differenoe i8 determined in a subtraotor 19. This difference is indioated as an analog signal by means of a dial gauge 20, but can also be transformed into a digital representation by means of an analog-digital-transformer.
Sinoe as a rule the analog signals given off by the photometer 12 for suooe~sive tests are of different values~ there should be an appropriate look, so that IV4(~ 0 a new output variable doss not appear in the output of the logarithmio analog-digital-transformer 13 until a constant absorbanoe value is indicated by the photo-meter 12 for a long period o~ time.
Since the test fluid, the solution of ad~uvants ; and the enzyme solution are continuously being conveyed~
i$ is simple to set up the device represented diagramma-tically in Fig. 1~ Only two periods need be measured empirioally for this:
a) the period from drawing in of the sample to appearance of the colored, reacted solution in the photometer. This period remains the ~ame when advance-ment of the material is ¢onstant~ and should be taken into oonsideration in ooordinating the mea~urement~
with the individual tests.
b) the period from the appearanoe of the oolored, reaoted solution to the reappearance of coloring of the solution flowing through the photometer due to a re-oiroulated enzyme solution, in the oase of oonstant advanoement of the ad~uvant solution and oonstant ad-vancement of water by means of the peristaltio pump 1.
The pulse frequenoy of the timing generator 15 should be set up in suoh a way that a determined measurement is advanced through all the levels of the slide register ~ust a~ter termination of this period.
In Fig. 2~ another devi¢e for performing the pro-oess o$ the invention i~ represented. A speoimen is advanoed from a oontainer 23 by a peristaltio pump 24 an ad~uvant solution is advanoed from a oontainer 25 by a peristaltio pump 26~ and a first-filling enzyme is advanoed ~rom a oontainer 27 by a peristaltic pump 28 through pipes 29~30 and 31 to a mixing point 32.

~ o The reaction mixture i~ then conduoted through a pipe 33 in which the reaction ta~es place~ to the inlet of the con~ntrate chamber 349 of a ~irst ultrafilter 36.
Through a pipe 37~ whioh is oonneoted to the filtrate chamber 35 of the ultrafilter 36~ the low-molecular oomponents of the reaotion mi~ture emerge and are ¢onducted to the inlet of the flow oell 38 of a speotrophotometer. Here, the light emitted by a lamp 39 up to a ¢ertain wave length is filtered~ and after passing through the flow cell 38 comes into oontact with a photo resistor 40~ whose resistance change is indioated by a measuring apparatus 41 either in the form of the light absorption or the extinction. The solution that issues from the flow cell 38 can be thrown away. A pipe 42 is oonnected with the outlet of the oonoentrate chamber 34 of the ultrafilter 36 The puri-fied enzyme is suotioned through this pipe by a pump 43 and at 44 it is mixed with a diluting agent pumped out of a oontainer 45 by a pump 46. The enzyme solution~
whioh is ~Ow again diluted to about the original con-oentration~ enters the conoentrate chamber 47 of the ultrafilter 49 and is pumped from the outlet of this ooncentrate ohamber 47 by a peristaltio pump 5~ through a pipe 52 to the mixing point 32 and mixed with the speoimen from the oontainer 23. The introduotion of the en~yme from the container 27 through the pipe 31 can be stopped by shutting down the pump 28 as soon as purified enzyme from the first test arrives at the mixing point 32 through the pipe 52. The low-molecular sub~tances that emerge from the filtratechamber 48 of the ~eoond ultrafilter 49 are conducted through a pipe 51 out of the filtrate ohamber 48 and oan be thrown awa~4~
In this mode of operation of the device Or the in-vention~ purified enzyme is mixed with new te~t material at the mixing point 32~ so that consideration oi the low-molecular component~ leit in the oon¢entrate in oalcu-lation~ ~rom pr3~ious determinations oan be omitted in thi~ ea~e. Figo 2 shows this mode of operation only diagra~matieally~ and it i9 equslly sel~-evident that t~e test material ~rom the test container 23 need not be the same in the case of several determination3 in a series, snd that a great number of similar containers 23 can be ~illed with different test specimens~ the content~
of which are advanced ~orward in a oertain sequence by the pump 24~ and the pipe 29 to the mixing point 32~
Moreover~ familiar devioes 08n be provided for separating the individual test epecimen~ such as, for example~
air bubble ~eparator~. More concentrate chambers and~
if neoessary~ more places 44~ in which diluting agents are mixed~ oan be added to the pipe 52, in addition to the concentrate ohamber 47 of the second ultrafilter 4g.
Exampless 1.) Determination of pyruvate with repeated uee of lactate dehydrogena~e Pyruvate may be determined by its enzy~atic reduoti~n to lactate, noting that an equivalent amount ; of NADH will be oxidized to NAD. ~he change oi the concentration in ~ADH will be photometrically measured at a wave len~th of 340 nm. The reaotion take~ place aocording to the equation pyruvate ~ NADH + H+ LD~ ~ lactate + NAD+
At a sufficient concentration of NAD~ (0~01 mMol) the ohemical equilibrium i~ totally at the right hand u~o s~de o~ the e~uation.
The apparatus ~as assembled accord~n~ to Fi~ure 2.
As the pumps 24, 26 and 28, peristallic pumps o~ the type "ISMATEC"*,mini were used toyether with a hose of a diameter of 0.8 mm and consisting o~ a plastics material "T~GON"*, said pumps having a supply capacit~ of 0.75 ml per minute. The pump 46 used was equally a pujp of the type "ISMATEC"* mini being equipped with a silicone hosè o~ a diameter of 1.5 mm and having a supply capacity of 2.6 ml pex minute. The pumps 43 and 50 - 10 were of the type KLB 4912A having a supply capacity of 0.2 ml per minute and being equipped with a silicone hose of a diameter of 1 mm, In a tengential stream housing having a diameter of 90 mm, filters of the t~pe "AMICON PM"* 30 were used (ultrafil-ters 36 and 49). The conduit 33 consisted of a polyethylene hose having a diameter of 1 mm and a length of 30 m and having a volume of 25 ml. All connecting conduits consisted of the same hose material~ , The connecting components used were screwing fittings consisting of "TEFLON"* and supplied by the firm Serva, Heidel-berg. The conduit 33 was located within an ultrathermostat according to Hoeppler and kept~ta temperature of 25C. The total volume of the enzyme circuit was 37 ml including the fil-trate portions. The photometer was of the type Zeiss PN2a with through-flow cuvette 38.
Reagents:
By dissolving 46.6 g triethanolamine.HCl in 200 ml water and adding 0.94 g complexon III and further adjusting the pH to 7.6 by means of 2N-NaOH and adding water for a total volume of 250 ml, a buffer solution having a pH of 7.6 was obtained. The solution of NADH

*,TRADEMARK

o wa~ u~ed in a 0.33 millimolar oonoentration within buffer solutionO The LDH was used in ~orm o~ a suspension of oristals as can be obtained on the market.
At the beginning o~ the determinations, the conduits 33~ 42 a~ 52 were ~illed with LD~ in bui~er solution. For this purpose the cristal suspension oontaining 300 pg LD~ uas diluted with 31 ml buifer ~olution and the solution obtained was sucked instead of the ~ample. As ~oon as the enzyme circuit had besn filled~ a ~olution of NADH and~ instead of the ~ample, water wa~ supplied a~d the zero level was read from the photometer. Subsequently~ the samples containing no protein~ and maximally 0.1 yMol pyruvate per millilitre were suoked. By separating air bubbles~ mixing of ad-~acent samples, which were supplied in time intervals of 60 seoonds~ was avoided. The extinction wlll decrease in proportion to the oontent in pyruvate aooording to the equation 0,~14 . E340 = ~pyr(pMol/ml) A oalibratlng ourve is not neoessary.
The enzyme solution remained fully active one week also with permanent use of the solution within the apparatus. With more extended worklng interval~ it is to reoommend to discharge the enzyme solution by opening a screwing connection and to store the ~olution at a temperature of 4C.

2.) Determination o~ D-gluoose with repeated use o~
the gluoose-oxydase and the peroxydase.
Glucose will be o~idized to gluoonic acid and hydrogen peroxide by GOD in the pre~ence of the oxygen oi~ ambient air. The POD will transfer the oxYgen o~

the H202 ~nto a chromogene and the extinction of the u~o oolouring matter formed will be determined. The react~ons take plaoe acoording to the equations:
~-D-gluco~e ~ ~2 ~ 2 - GOD ~ D-gluconic aoid ~ H202 ~2 + D~2 ~ ~2 POD ~ 2 H20 ~ D

Both chemi¢al equilibrla are totally shifted to the right hand side-of the above equations.
The apparatus used wa9 assembled in an analogous ma~ner to the apparatus described in 1.). In partioular the same pumps and the same oonnecting conduits were used. However, the ultrafilters 36 and 49 used were of the Amicon type PM 10. The ultrathermo~tat according to Hoeppler was ad~usted to 35C in thi~ case.
Reagents:
A buffer solution having a p~ of 7 was prepared by dig801ving 2.76 g Na2HP04.2~20 and 1.45 g Na~2P04.2~20 in iOO ml water. In this oase~ 100 mg ABTS (2~2~-azino-di-(3-ethylbenzthiazolon)-6-sulfonate) was used as the ohromogene in 100 ml buffer solution. ~he gluoose o~ida~e as well as the pero~idase were used in solid form (as oan be obtained on the m~rket)~ dissolved in buf~er solutio~.
At the beginning of the determinations~ the conduits 33, 42 and 52 ~ere filled with GOD and POD in buffer solution. For this purpose 4 mg GOD and 1.5 mg POD were dissolved in 31 ml buffer solution and this solution was suoked instead of the sample~ thereby sucking~ instead of a solution of ohromogene, pure buffer ~olution. As soon as the enzyme circuit had been filled~ the solution of the chromogene was supplied and a calibrating ourve was established by means of a plurality of standard solution~ of glucose o containing 1 to 20 pg glucose per milliliter. Sub-sequently tha sampla~, whioh did not contain proteins and oontained maximally 20 pg gluoose per milliliter~
were s~pplied. By separating aIr bubbles~ intermixing of adjacent samples wa~ avoided, the samples being ~upplied with a time interval of 60 seoonds. The extinotion is proportional to the content in gluoose.
Mea~urements were taken at a wave length of 420 nm and tran~formed by means of the oalibrating ourve into the ~ought result~.
Thè enzyme solution remained fully active ona week al~o with permanent use of the solution within the apparatus. During working intervals it is to reoommend to discharge the enzyme solution by opening a screwing oonneotion and to store the solution at a temperature of l~C

-17- 11.7.7~/li

Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of determining the concentration of low-molecular weight biological substances by enzymatic reaction, wherein the low-molecular weight reaction products obtained as a result of the enzymatic reaction together with the low-molecular weight biological substance are separated from the enzyme after the enzymatic reaction by ultrafiltration, and wherein the enzyme used therein, which emerges as a concentrate from the ultrafilter separately from the reaction mixture, is recycled and used again for enzymatic reaction with low-molecu-lar weight biological substances, the concentration of the bio-logical substances being determined by means of the measured concentration of the low-molecular weight reaction products.

2. A method as claimed in claim 1, wherein low molecular weight substances left in the concentrate after the enzymatic reaction and after the ultrafiltration are determined on the basis of the concentration of the reaction products measured after the enzymatic reaction, and are considered in calculations during the next determination using the enzyme remaining in the concentrate.

3. A method as claimed in claim 1, wherein the concentrate flowing from the concentrate chamber of the ultrafilter is diluted and ultrafiltered again, and wherein the concentrate from the last ultrafiltration is used again for enzymatic reac-tion with low-molecular weight biological substances.

4. In a method for determining the concentration of low-molecular biological substances by reaction with an enzyme including the steps of contacting a low-molecular biological sample with an enzyme, thereby producing low-molecular reaction products, quantitatively measuring the concentration of the low-molecular reaction products and discarding the unreacted sample and the low-molecular reaction products after measuring, the improvement of recovering a substantial portion of the enzyme comprising separating the low-molecular reaction products and unreacted sample from the enzyme by a first ultra-filtration into a filtrate containing substantially all of the low-molecular reaction products and unreacted sample and a concentrate con-taining the enzyme and a trace quantity of the low-molecular re-action products and unreacted sample, diluting the concentrate and subjecting the concentrate to at least one additional ultra-filtration, recovering the enzyme therefrom substantially de-void of low-molecular reaction products and unreacted sample, and reusing the thus separated enzyme in the continuous concen-tration determinations.

5. A method according to claim 1 or 4 wherein the recov-ered enzyme is stored prior to reuse.

6. The method according to claim 1 or 4 wherein the quan-titative measurement of the concentration of the low-molecular reaction products is effected the filtrate separated from the first ultrafiltration step, and the concentration of the sample as calculated from the measured concentration of the low-molecu-lar reaction products.

7. An apparatus for quantitatively determining the concen-tration of low-molecular biological substances by reaction with an enzyme and production of low-molecular reaction products including:

a source of low-molecular biological sample;
a source of reactive enzyme;
pump means for delivering a predetermined quantity of sample from the sample source to a reaction area;
pump means for delivering a predetermined quantity of reactive enzyme from the enzyme source to a reaction area;
means for mixing together the delivered sample and the delivered enzyme including therewith a reaction area;
pipe means communicating the reaction area to a first ultrafilter for separating a filtrate containing substantially all of the low molecular reaction products and unreacted sample from a concentrate containing the enzyme and a trace quantity of the same and low-molecular reaction products;
a second ultra-filter for further concentrating the enzyme and removing trace portions of sample and low-molecular reaction products therefrom;
pipe means communicating the concentrate of the first ultra filter to a second ultra-filter;
a source of enzyme diluent intermediate the first and second ultra-filters for delivery of enzyme diluent to the concentrate passing from the first ultra-filter to the second ultra-filter.
pipe means communicated from the outlet of the second ultra-filter for delivering theseparated, diluted enzyme substantially devoid of sample to the mixing means;

pipe means communicating the filtrate of the first ultra-filter to an outlet for discarding the un-reacted low-molecular sample and the low-molecular reaction products, and means for determining the concentration of the low-molecular reaction products for evaluation of the concentration of the low-molecular biological sample.

8. The apparatus according to claim 7 further including a third ultra-filter for further concentrating the enzyme, said filter connected to the second ultra-filter via a communicating pipe therebetween for delivering filtered concentrate to the third ultra-filter;
a second source of enzyme diluent associated with the third ultra-filter intermediate the second and third ultra-filters for delivery of enzyme diluent to the concentrate passing from the second ultra-filter to the third ultra-filter, and pipe means communicated from only the outlet of the third ultra-filter for delivery of the diluted, separated enzyme substantially completely devoid of sample to the mixing means, the three ultra-filters connected in series.

9. The apparatus according to claim 8 further including:
storage means for the separated enzyme intermediate the pipe means from the third ultra-filter and the mixing means.

10. The apparatus according to claim 7 wherein the said means for determining the concentration of the low-molecular re-action products is arranged in the said pipe means communicating the filtrate of the first ultra-filter to an outlet for discard-ing the unreacted low-molecular sample and the low-molecular reaction products.