CA2233701A1 - Analysis method - Google Patents

Abstract

A method for determining the activity of a nucleotide polymerizing enzyme in a sample, and use of the method for determining HIV 1 RT- and Herpes Simplex DNApolymerase activity. The enzyme is captured by means of a monoclonal antibody which is immobilized to a solid carrier and is capable of binding the enzyme without detrimentally effecting the enzyme activity. Contaminants and disturbing factors are removed and the nucleotide polymerization starts by the addition of a reaction solution containing a primer/template construct and nucleotides substrate, the reaction conditions being chosen such that they promote permanent association between antibody enzyme- and primer/template constructs. When necessary nucleotide substrate, primer/template and reaction solution are washed away from the newly synthesized polymer, and the amount of nucleotide which has been incorporated into the polymer is determined, and the activity of the enzyme is determined with the guidance of this determination.

Description

MET~OD FOR DETERMlNlNG ACIlVllY OF REVERS~ TRANSCRllrTASE
The yresent-invention relates to a method for determining the activity of certain DN~ polymerizing enzymes in human or animal body liquidsI cell samples or samples from virus infected cultures, these enzymes being caracterized by high yrocessivity. The invention also relates to the use of said method for isoenzyme-a typing. The invention also relates to use of said methods for diagnosis and prognosis of tumour deseases or deseases which are caused by or associated with virus infections such as AIDS, T cell leukaemia and herpes virusinfections Background of the invention Reversed transcriptase, in the following called RT, is a critical enzyme 10 ~hich jc res}~onsable for the synthesis of DNA from viral RNA for all retro-v iruses, inclucling human immune deficiency virus (HI~'). Three different enzymatic acti~,ities are involved in this process: synthesis of the ~lrst DNA
stran(l, degradation of the viral RNA strand in the DNAJRNA hybride and synthesis of the second DNA strand. See e.g. S.P. Goff, (199Oj Review, J, AIDS 3, yages 817-831 for an overview.
The RN~ and DNA dependent polymerase activities are obviously mediated by the same catalytical grouy, ~vhereas RNas H activity is mediated through a special yart of the molecule. Se e.g. J. Hansen et al. EMBO J. (1988) 7, pages 239-243, M.S. Johnson et al. (1986) PNAS 83, pages 7648-7652. HIV 1 RT appears as 20 a heterodimer in virions consisting of two polypeptides, p66 and pal, with the same ~ terminals Isee e.g. M.M. Lightfoot et al. (1986)J. Virol.60, pages ~ 71-77 a, Veronese et al. (1986) Science 231, yages 1289-l29ll p51 is generated by viral protease cleavage of the p66-polypeptides in the dimer of p51 and pla (see e.g.
Mous) ~n the heterodimer p66 catalyses the polyemerase reaction, but the same 2a sequence in p~1 does not Isee e.g. Le Grice et al. (1991) EMBO J. 10, pages 3905-3911, Hostomsl~y et al. ~1992), Science 256, pages 1783-17901. It is assumed that p~1 is a part of the binding site for the tRNA primer and a part of the template-primer bincling site (see e.~. Kohlstaedt 1992~ The RNA-ase H peptid activity inthe p66ip51 heterodimer is locaiized to the C terminal part of p66 (see e.g. Hansen 30 198~).
Analysis relating to RT acti~rity has become an accepted technic for detectioning and quantification of retrovirus in cell cultures (see e.g. Pioesz 1980 and Barr-~iinoussi 1983). Together with the p24 antigen test it is used as a confirming test for HIV isolation (see e.g. Jackson 198~, Gupta 1987). RT is also 35 the main target in attemyts to find efficient anti-virales against HIV. ~lany attempts have been made to find selective inhibitors of this enzyme. Although nocure for A~DS not yet has been found, a valuable effect is achieved by treating patients with 3'-azido-3'-deoxy thymidine (AZT). It has, however, been found that treatment with AZT only in a rather short period of treatment (from 3 months to 5 1 year) produces therapy resistant HIV, with mutated RT. RT tests are presently used for evaluation of reaction mechanisms in potential antivirals. Another great yotential use of RT analysis is consequently characterisation of enzymes from therapy resistant mutating viruses.
Conventional measurement of RT activity is carried out ~vith the enzyme 10 in solution, an artificial templatelprimer construct and tritiated deoxynucleoid triphosphate as the nucleotide substrate (see e.g. ~3altimore 1971, ~oon & Lee).This system is based on detecting incorporation of radio activity in RN~/DNA
hybrides which can be precipitated with trichloro acetic acid (TCA). By using ,~emitting nucleotides scintillation liquids can be used for detecting radio activity, la but this often results in poor reproducability depending on eYtinction problems.
The stanAard test is comparatibly labor consuming and can not readily be adaptedto large scale investigation of a large number of samples. It is also very sensitive to the effects of disturbing factors in the enzyme samples. The latter are often the critical factor for the determination of RT activity in extracts from infected cell 20 cultures or HIV infected individuals. Furthermore, the activity of cellular gamma~olymerase enzyme is a potential specificity problem.
During recent years the intensive research relating to HIV has resulted in improvements of RT tests by the use of various techniques.
The incorporation of 125I labelled substrate resulted in improved sensitivity 2a and eliminated quenching and use of scintillation liquids (Gronowitz et al. 1g90).
The introduction of templates or primers coupled to solid phases simylified the separation between substrate and product, simplified the need of precipitation with TCA and resulted in a "one tube RT test" (see e.g. Gronowitz 19g1, Urabe et al. 1992). Several attempts have also been made to make the test systems less30 sensitive to disturbing factors.
According to Porstmann et al. 1991 a monoclonal antibody against HIV1-RT is used, ~vhich binds to a well in a microplate in order to isolate RT before the analysis. However, this method has been used for determining the RT activity in virus samples which already have been purified by precipitation with PEG and 35 subsequently centrifugation. Furthermore, after immobilization of RT, a comple~c procedure is used for separating the nucleoside substrate and the soluble labled WO 98/06873 PCTtSE96/00990 product. Grono~,vitz et al. 1991 uses prA/odT bound to a plastic carrier, for affinity purification of HIV-RT in one step directly from cell e~ctracts. The same prAlodT
construct is then used as primer/template in the subsequent RT test step.
Recently it has been avoided to use radio activity as a marker in RT
5 analysis by instead using modified nucleotide bases cont~ ing antigenic epitopes or structures having av high affinity for defined ligands. The presence of theseepitopes or structures in the recently synthesized RNAJDNA hybride is then used for binding antibodies or ligands which have been conjugated with Eg ELISA
enzymes. The amount of bound ELISA enzymes has then been determined with 10 a secondary enzym test. Porstmann et al., 1991, makes use of 5-bromo-deoxy-uridine (~rdU) triphosphate as the nucleotide substrate in RT analysis. The amount of incorporated BrdU is determined in a second step with an immune test while using monoclonal anti-BrdU antibodies.
Beery & Scieb, 1992 measures the incorporation of dio:cygenine-lablecl 15 dUTP in newly synthesized DNA instead of radioactively labelled dTTP. In order to be able to separate the non-incorporated nucleotides from newly synthesized DNA also biotine-labelled dUTP is added to the reaction mi~cture. After reversedtranscriytion the newly synthesized double labelled DNA is immobilized on strept-avidine coated ELISA wells and determined photometrical by binding of peroxy-20 dase-conjugated anti-digoxygenine antibodies. This method has been the basis for an RT test kit which is available from Boehringer, Mannheim.
Urabe et al. has developed a non-radioactive RT test ~,vhich is based on the incorporation of biotine-dUTP in an immobilized odT/prA construct. The amount of incorporated nucleotide substrate is measured photometrical after addition of25 strept-avidine conjugated alkaline phosphatase ~AP).
Another commersially used principle for showing RT activity is to use a series of specific sonds for detecting newly synthesized cDNA. This enzymatic reaction makes use of a heteropolymer RNA molecule with a 20 bases oligonuc-leotide-primer which is complementary with the RNA sequences close to the 5' 30 end. During the RT reaction a complete cDNA strand is produced. After hydro-lysis of template-R~A, cDNA is hybridised ~vith t~,vo different oligonucleotide sonds, the capture and detection sonds respectively. The capture sond is used for binding cDNA to wells in a microplate. The detection sond is conjugated to horse-radish pero:Yidase, which results in a colour reaction after washing for removing 35 unused nucleotide substrates and free sonds.
One purpose of the present invention is to provide a method for quantita-tive determination of certain polymerase activities which removes most of drawbacks of the previously known methods. Another purpose is to provide a kit for specific determination of different polymerase activities. The invention also relates to the use of said methods and products for novel purposes.
These and other objects of the invention, and how they are achieved, will be explained and illustrated in further detail in the subsequent description andthe working e~camples.
The invention is especially illustrated in connection with determination of HIV1 RT- and Herpes Simplex DNA-polymerase activity, but it can also be used in all highly processive polymerases such as retro RT, DNA virus polymerases, cellular gamma- and delta-polymerases and the like.

S--mm:~ry of the invention One yurpose of the present invention is to provide methods, means and devices for diagnosis and supervision of certain diseases which are related to retrovirus infections.
Another object of the invention is to provide a sensitive test system which is capable of detecting minute amounts of retroviral RT in body liquids from humans or animals, cell samples or samples from virus propagation cultures, saidsystem also being constructed so as to minimize the effects of the disturbing factors which are present in the above mentioned types of samples.
A still further object of the invention is to provide a method for eliminating the ef~ect of the cellular polymerases, e.g. gamma polymerase, which may cause unsuitably high background activity in sensitive RT tests.
A still further object of the invention is to detect other nucleotide polymeri-s}ng enzymes tvhich are characterized by high processivity, which is used according to the invention.
The above and other objects of the invention will be e~{plained in more detail in the following description of the determination method and its appli-cations according to the invention.
The invention thus provides an improved analysis system for certain nucleotide polymerising enzymes. It is characterized by 1) using, in an initial capture ste~, a monoclonal antibody which is immo-bilized on a solid phase and is capable of binding at least one enzyme without detrimentally effecting the enzyme activity, 3~ 2) removing impurities and disturbing factors in a subsequent step, preferably - by washing them away, 3) starting the reaction by adding a reaction solution which contains a primer/template construct, nucleotide substrate and preferably essential salts and co-factors, the reaction conditions being choosen so as to favour permanent 5 association between the antibody-enzyme- and primer/template-constructs, 4) if necessary washing away untreated nucleotide substrate, primer/template and reaction solution and 5) determining, in a manner which is known per se, the amount of nucleotides which have been incorporated into the newly synthesised polymer.
This determination can e.g. be carried out by radioactive labelling, in which case the measurement can take place directly, or e.g. have the form of different types of modified bases, in which case the determination can be done by secondary reactions.
The key feature of the invention is step 3. Provided that suitable reaction conditions are used, the enzymes will bind strongly to the yrimeritemplate construct and the new labelled strand which is produced will remain bound to theimmobilized enzyme. This makes it possible to separate substrate from product using a siml~le washing step, as mentioned above.

Short description ofthe dra ~ ngs Figure 1 shows the amount of' RT activity which has been recovered on l~~ab beads as a function of absorption time and temperature.
Figure 2 is a diagram which sho~qs the template/primer dependence (prAlodT) for optimal RT activity and linearity in time.
Figure 3 shows the linearity with time and the amount of enzyme for the RT capture test.
Figure 4 shows a comparison between the substrate kinetics for free and immobilized HIV 1 RT respectively.
Figure 5 is a diagram which sho~vs the distribution of the RT reaction product between ~/Iab H2 beads and reaction solution.
F;gure 6 is a diagram which ill~lstrates processive DNA synthesis over template boarders in the RT capture test.
Figure 7 is a comparison between the effects of Iymphocyte e~ctract on RT
capture test and soluble test respectively.
Figure 8 is a diagram which compares two product detection systems in the RT capture test.

Figure 9 shows the use of a capture test for measuring Herpes Simplex type I DNA polymerase, the various symbols representing four different Mab.

S-lmm~ry of the tests illustrated in the drawings The results according to Figure 1 were obtained by incubating HIV 1 RT
5 l2,7x10-15 moles~ at 8~C ([~) or at 20~C (--) with Mab H2 bound to beads. The binding buffer was removed from the Mab beads at the indicated times and the distribution of RT between Mab beads and binding buffer was determined.

A) RT capture test: The Mab beads were washed and the RT activity which had been bound to the Mab beads was determined by an RT reaction for 2h using 10 1,5x10-7M of 3H-TTP (60 Ci/mmoles) as dNTP substrate. The reported data relate to cym which have actually been recovered on each bead.

B) Soluble RT test (Prior art): Remaining RT activity in the binding buffer was determined at the indicated times using a conventional RT test with 1,5x10-7M
of 3H-TTP (60 Cilmmoles) as dNTP substrate. The RT activity was recalculated 15 as yrocentage of a control consisting of RT in binding buffer which had been stored at 8~C without any addition of Mab beads.
In the test which is illustrated in Figure 2 ~IV-1 RT (1,8x10-15 moleslsample) was incubated with Mab H2-beads for 3h at 20~C. The Mab beads ~,vere then washed and reaction solutions were added l,vhich contained 1,5x10-7M20 of 3H-TPP (78 Ci/mmoles) and varying concentrations of prA300 and odT20. The reaction solutions were incubated at 33~C and samples were taken after 2 (--), 4(Cl) and 16 (--) h respectively.

A) The amount of reaction product which had been recovered from the reaction solutions containing 20 mg of prA/ml and the indicated amount of odT20.

2a B) The amount of reaction product which had been recovered from reaction solutions containing the indicated prA concentration when using a constant relation for prA/odT of 100/1.
The results which are shown in Figure 3 relate to series dilution of HIV-1 RT from 2.5x10-16 to 2.6x10 13 moles of enzyme per sample, incubated with Mab 30 H2 beads for 3h at 20~C. The MAB beads were washed and a reaction solution containing 1.5x10 7M of 3H-TPP (78 Ci/mmoles) was added. The reaction solutions WO g8/06873 ' PCT/SE96100990 were incubated at 33~C and samples were taken out at the indicated time.

A) Symbols (moles of enzyme per sample): 2.5x10-16 (O), 1.0x10-15 (--), 4.0x10-1~ (O), 1.6x10-14 (O, 6.4x10 14 (O and 2.6~10-13 (-).

B) Symbols (time in hours): 2 (--), 4 (C), 16 (-) and 32 (O).
Figure 4 shows initial reaction velocities at varying substrate concentra-tions for HIV-1 RT which has been bound to Mab H2 beads (-) and for free HIV-1 RT (o). The values have been obtained by measuring the reaction velocity for three different amounts of enzyme (1.28x10-14 moleslsample, 2.56x16-4 moles/sample and 4.52x10-14 moles/sample) and five different substrate concentra-tions (from 1.1x10 7M to 3.2x10-5M) using the capture test and the conventional RT test res~ectively. The test data obtained have been recalculated to the same amount of enzyme (4.52x10-4 moles/sample).
The diagram in Figure 5 shows the distribution of labelled freshly produced DNA between Mab H2 bead and the reaction solution after polymerisation reaction. The measurements have been carried out for three different enzyme concentrations (see Figure 4) and five substrate concentrations (see Figure 4). The samples which were taken after 2, 4 and 18h respectively. Samples which did not give linearity in the RT capture test, due to depletion of the nucleoside substrate, were included in the analysis, whereas samples which did not provide significant20 detection (less than three times the background in the capture test) were exclu-ded.
In the test which is illustrated in the diagram in Figure 6, HIV-1 RT
(4.6x10-15 moles/sample) were at fir~t incubated with Mab H2 beads for 3h at 20~C, unbound enzyme was removed by washing of the beads and the enzyme 25 reaction was started by addition of a complete reaction solution containing 7.1x10-7M of TTP (12.1 Cilmmoles), prA (20 mglml) and odT (200 nglml). The amount of available A-bases on each bead, when all bound enzyme molecules bind one template (300 bases), corresponds to 8.2x10 l3 moles.

A) The first reaction solution, containing free prA/odT, was removed from the 30 beads after 2h of incubation (at 33~C) and was then replaced by new solutionscontaining 3H-TTP and the indicated additional components, prAlodT (--), prA
(--), odT (O), none (O) and no pr~VodT respectively between 2 and 4h, and prA/odT was added after 4h of incubation (~). The samples were taken out at the WO 98/06873 PCr/SE96/00990 - indicated time and were re~calculated to the number of mo}es of TMP which had been incorporated in to DNA.

B) The first reaction solution was removed from the beads after incubation for lh and was replaced by new reaction solutions cont~ining prAlodT and either 5 unlabled TTP (O, ~) or 1.8x~0-5M ddTT ( O, ~ ) as trinucleoside substrate. These reaction solutions were removed after incubation for another 2h (not shown on the x scale in the figure) and were replaced by new solutions Cont~ining 3H TTP a~rdeither prA (O, O) or odT/prA (--, ~) in standard concentrations. The samples were taken at the indicated times and were recalculated into the number of moles10 of TMP that had been incorporated.
The results which are reported in the diagram in Figure 7 were obtained by mixing of recombinant HIV-1 RT (2.5x10-l4 moles/sample) with the indicated amount of e~tract from peripheral blood lymphocytes and the RT activity in each - sample was determined by RT capture test and soluble RT test respectively. The symbols have the followingme~nings: standard capture test (--), capture test with 2 mM phenylmethyl-sulphonylchloride (PMSF) and 20 g of prA included during the enzyme binding step (O), soluble standard test (O), soluble test with 2 mM
of PMSF added to the reaction solution (--).
Figure 8 shows how three dilutions of HIV-1 RT (6.4x10-16 moles/samyle, 6.4x10-l5 moles/sample and 6.4x10-14 moles/sample) were incubated with mab H2 beads for 3h at 20~C. The amount of RT which was bound for each enzym dilution was determined by the use of two RT test systems. One of them was a standard test with ~.5x3-7M of 3H-TTP as nucleoside substrate and direct detection of incorporated 3H-TMP. The second test ~,vas carried out using BrdUTP as nucleo-side substrate and immunological detection of the reaction product. In both cases an incubation period of 150 minutes at 33~C was used for the polymerization step.
Incubation at 20~C for 50 minutes was used for the Ap reaction.

Sllmm~rv of the tests reported in the Tables Table 1. Characterization and selection of Mab for the RT capture test Beads with immobilized monoclonals were incubated at 20~C for 2h with recombinant HIV-1 RT in binding buffer -/+0.5 M KCl. The amount of captured RT on Mab beads is reported as 3H-TMP which have been incolporated in bound and free nucleic acids respectively. Remaining RT activity in binding buffer without salt was determined by using a soluble RT test and was recalculated to 3 P~ OO990 - '~c of a control consisting of HIV-1 RT which had been incubated with the non-HIV
beads. The cayability of each monoclonal to inhibit RT activity was checked in aseparate test. Each ascites fluid was incubated with recombinant HIV-1 RT.
Remaining RT activity was determined by the use of soluble RT test and recalcu-5 lated to Yc of HIV-1 RT which had been incorporated with non-HIV ascites.

Table 2. The linearity with time in the presence of cell extracts The indicated amounts of recombinant HIV-1 RT were mixed with different amounts of an extract from peripheral blood Iymphocytes. The recovery of RT
activity was determined by means of the RT capture test and the correlation 10 coefficients between the amount of product formed and the time of test was calculated. The amount of formed product ~,vas also re-calculated into 5'c of the recovery from a control containing the corresponding amount of RT but no cell extract.

Table 3. The linearity with the amount of enzyme in the presence of cell extract A set of seven different 4-fold dilutions of recombinant HIV-1 RT ~from 7.81x10-17 to 8.00x10-14 moleslsample) were mixed with an extract from 1.00x10-6peripheral blood Iymyhocytes (PBL). The recovery of RT activity was determined by means of the RT capture test and correlation coefficients between the amount 20 of used enzyme and the amoun~ of f'ormed product was calculated directly fromthe cpm values obtained. For the used minimum, median and maximum concent-rations of the enzymes the product recovery was also re-calculated into 5'c of the recoveIy of a control which contained a corresponding amount of RT but no cell extract.

25 Table 4. The capabi~ity of the capture test to recover HIV-l RT from various types of samples Various components such as anticoagulants, Ficol, a protease inhibitor (PMSF) and cell e~ctract were added to the binding buffer. The concentrations ofthe anticoagulants were those which are used for routine plasma sampling. The 30 concentration of Ficol was 505~ of the total binding buffer, and 2 mM of PMSFand 20 ,ug of prA were used as protecting agents. The beads were incubated for 3h at 3.3~C with HIV-1 RT and the various comyonents were washed and the recovered RT activity ~vas determined in a 2.5h test. The data obtained were - recalculated as ~c of an identically treated control, without any additives to the binding buffer. The upper part of the table shows the effect of the indicated comyonents in the absence or presence of e~tracts from washed whole blood cells which had been isolated from citrate blood and re-suspended in l~inding buffer to 5 half of the concentration in the original blood. The lower part shows the effect of including the indicated type of cell extract in the binding buffer.

Working e~amples The herein described RT capture test makes it possible to directly measure or detect RT also in impure samples and simplifies the final separation of 10 substrate and product. The test comprises three main steps: first immunoaffinity purification of RT, then polymerization reaction and finally separation of sub-strate and product.

Selection of RT bintlin~ monoclonals and binding conditions for the immunopurification step The well known problems with measurement of DNA polymerizing enzymes in impure biological samples are the starting point for the problems which the present invention are intended to solve. The original idea was to construct a system for removing HIV RT from any unfavourable invironment by cayturing the enzyme with use of an immobilized antibody against the enzyme. Disturbing 20 factors such as RNA-ses, DNA-ses, nucleotidases, proteases and competitive templates/primers could then be removed simply by a simple wash of the anti-body-carrier. The optimal antibodies for this purpose should have the ability torapidly capture RT in crude samples from all known HIV-1 isolates. They should also be capable filling this function without determentaly effecting the polyme-25 rizing activity of RT. Several research teams have produced panels of monoclonalsagainst HIV RT (Orvell et al. 1989, Restle et al. 1992, Szilway et al. 1992). A set of 18 mab were screened regarding the ability of rapidly capturing RT from HIV-1in crude samples and interference with the activity of the captured RT enzyme.
The various Mab clones were reactive with seven different epitopes of the HIV-1 30 RT protein (Table 1, column II).
The total recovery of radioactive reaction product in the capture test (Table 1, columns IV-VII) is a function both of the binding efficiency of the indicatedMab and the final activity of the bound enzyme. The capability of each Mab to capture RT can be seen from Table 1, column III, which shows r~m~ini WO 98/06873 PCT/SE96/OOg90 - unbound RT activity in the binding buffer after incubation with the various Mab beads. The effect of the binding of each free Mab to HIV 1 RT in solution was evaluated in a separate RT inhibition test (Table 1: VIII). The ~Iab bead against the epitope 1, 4, 5 and 6 did not bind RT in a satisfactory manner. Mab beads against epitope 3 could bind RT, but the bound enzyme did not have the ability to efficiently synthesize DNA product due to partial inhibition of the RT activity (Table 1: VII~). Two Mab against two different t"arts of HIV 1 RT, H2 against p51 and Q7 ~gainst pl5 (Orvell et al. 1989) were found be suitable for our purpose and were selected for further studies. These Mab were also found to be non-competi-tive and provided an additative effect in the capture test. A com~ination of H2 and Q7 on each bead was used for analysis of clinical material.
The proportion of RT which was recovered on the Mab beads was also dependent on the absorption time and temperature which ~vas used in the binding step, as can be seen in Figure 1. RT was incubated with the ~Iab H2 bead for theindicated time and at two different temperatures. The amount of RT activity on the bead increased with the incubation time, resulting in a proportional reduction of the remaining unbound RT activity in the binding buffer (Figure 1).

The polymerization reaction's dependence of primer and template in the capture test The relation between odT primer concentration, reaction velocity and linearity in time on Mab H2 beads is e~cemplified in Figure 2A. The primer requirement showed a rather wide optimum and the reaction velocity reached a m~rimum at 10-1000 ng odT/ml which corresponds to 0.1-10 odT 20 primersi-template. Use of smaller amounts of primer resulted in reduced reaction velocityand also poor linearity vs time. If the reaction solution completele lacked primer there was only formed an insignificantly detectable reaction (Figure 2A).
The reaction velocity as a function of the template concentration at a constant prAlodT relation of 100:1 is shown in Figure 2B. A saturated system in which the template concentration did not limit the reaction velocity anA linearity with time was reached when using at least 20 mg prA/ml, corresl~onding to 1.08x10 8 moles of A bases/tube. The combination of 20 mg/ml of prA and 0.2 mglml of odT20 showed to be suitable as standard conditions for our RT capture test.

WO 98/06873 PcT/sE96/oosso - Evaluation of the enzymatic properties of the immobilized HIV 1 RT in the capture test The relation between the amount of formed product and the reaction time for RT is illustrated in Figure 3A. The enzyme reaction ~,vas linear at least up to 16h as long as not more than 1.6X10-14 moles of ~IIV 1 RT ~vas used in each test.
Higher enzyme concentrations result in substrate depletion, thereby shortening the time for linear enzyme reaction. When using an analysis time which is shorter than 4h and using 1.5x10 7 M of 3H-TTP (80 Ci/mmoles) as substrate, a linear relation is obser~ed between incorporated marker and amount of enzyme, up to 6.4~10-l4 moles of HIV 1 RT in each test (Figure 3B). Figure 4 shows a Menten diagram which illustrates the nucleotide substrate kinetics for free and immobilized HIV 1 RT. The initial reaction velocities for five concentrations ofTTP, from 1.1x10 7 to 3.2.Y~0 5, were determined by means of a conventional solub}e RT test and with the RT capture test according to the invention, identical reaction conditions being used. Free HIV 1 RT in soluble test resulted in a Km value of 1.21~10-6 M, ~,vhich can be compared ~vith 1.18xlO-6 M for HIV 1 RT
which has been immobilized on mab 2 beads. When the reaction velocities are recalculated into equal amounts of RT added to both test systems (4.52~c10 14 moles/sample, Figure 7), free and immobilized enzyme had Vsat values of 2.04x10-9 and 1.18~c10-9 M/min respectively, i.e. the Vsat value seems to be about 40~c lo~,ver for the immobilized enzyme. It should, ho~vever, be kept in mind that the comparison of the reaction ~elocities according to Figure 4 is based on additionof equal amounts of RT in each test. The binding of RT to immobilized H2-mab is a time and temperature dependent reaction. As appears from the diagram in Figure 2, about 40C~c of total RT ~vhich has been added to the ~ample will remain in solution under the binding conditions which are described in connection with Figure 6 (0.5 M ~Cl, 20~C and an incubation time of 3h). The nucleoside sub-strate kinetics will obviously not be essentially changed by the immobilization of the enzyme. This means that the RT capture test according to the invention can be used for characterization of possible antiviral substances.

Separation of substrate and product in the RT capture test In the last step of the RT capture test rem~iningTTP substrate from TMP, which has been incorporated into the DNA product, ~vill be separated by a simplewash of the carrier bead. Not only the substrate but also all DNA product ~,vhich has been released from the immobilized enzyme during the reaction will then not WO 9~/06873 PCT/SE96/00990 be detected. The effect of these factors on the product recovery was determined by analysis of the distribution of the RT reaction product between Mab H2 beads andthe reaction solution within a broad range of products amounts, as illustrated in Figure 5.
As can be seen from this Figure, theses variables were correlated (r = 0.46, m = 4.5, p < 0.005). Increasing the amount of bound product resulted in an increased amount in the free product. The found relation between free and bound reaction product was between 2x10 4 and 1.7x10-2 (median value 2.3x10-3).
However, the amount of free labled nucleic acid was never greater than 1.7~c of bound nucleic acid, in spite more than 250 fold variation of the amount o~ product formed.

P~ocessive DNA synthesis over template boarders in the RT capture test One of the more surprising properties of the enzyme test according to the invention is that virtually all product from the enzyme reaction is recovered asimmobilized enzymelproduct complex. Release of the template from the immobi-lized enzyme obviously a very rare occation once the elongation reaction has started. When starting on a primed, about 300 A bases long template, the RT
capture test according to the invention is capable of providing a linear incorpo-ration of 3H-TTP for at least 32h. A calculation of the total amount of the immobilized T strands which are produced for 16h using 4.6x10-15 moles of HIV
1 RT/sample1 provides about 11,000 bases. It is true that the processivity of HIV-1 RT on prA templates is unusually high (Majumdar et al. 1988, Huber et al. 1989),but the product is more than 30 times longer than the template used.
In the test illustrated in Figure 6 there is at first created an immobilized enzyme-primer/template-complex under 3 hours of incubation at 20~C. The effect of the addition of reaction components is then evaluated for a second incubationperiod. The amount of incorporated TMP product per molecule of bound enzyme was in long term tests (16h) for the r eactions with excess of prA found to e~ceed the amo~mt which is possible to incorporate in a single template (300 A bases), more than 30 fold. Removable of the temylate from the reaction solution reduced the reaction velocity dramatically, ~,vhereas addition of a ne~v template restored the velocity (Figure 7A). The enzyme reaction obviously uses free A strands as templates for the growing, immobilized pdT strand. A possible explanation of these observations is that the reaction starts from the initial primer, proceeds CA 0223370l l998-04-Ol WO 98/06873 P~ ih~ 00990 along the first template stretch to the template limit. The end of a new prA
template is then incorporated into the growing hybride and the enzyme reaction will continue. This sequence will then be repeated for the ne~t template-boarderand so on "eternally''.
A confirmation of this idea is that it has been reported (Hubert et al. 1989) that HiV-1 RT is capable of forming long projecting single stranded DNA strands over the template boarders. The same scientists also have hinted that the enzymeremained bound to the end of the extended primer.
The capability of the immobilized enzyme to change primer ~vas investi-gated in a special test. An immobilized enzyme template/primer-complex was produced during a first incubation. This extended primer is made useless by a second incubation with 1.75~c10-5 M of ddTTP. The effect of addition of a new primerltemplate was then, after removal of excess of ddTTP, controlled by l,vashing of the immobilizecl enzyme complex. The results showed that virgually all activity of the immobilized enzyme had disappeared and not could be restoredby adding ne~,v prA or pr~/odT (Figure 6B).
Taken all together our data show unexpectally high yrocessivity for the RT
enzyme. Separation of substrate and product is simple. Just wash the beads. Thishas made it possible by optimation of the analysis conditions in order to avoid dissociation of the enzyme-primer/template-complex Important factors are the useof a prA template, low ion strength and incorporation of an ~Nas ~ inhibitor (e.g.
dextrane sulyhate) in the reaction solution.
Comparison between the RT capture test and soluble RT test in the presence of cell extract Recombinant HIV-1 RT was mixed with the indicated amounts of an extract of periyheral blood lymphocytes and remaining RT activity was deter-mined ~,vith the RT capture test according to the invention and soluble RT test respectively. The amount of cell extract which inhibited 50~c of the RT activitycorresponds to about 10,000 cells in the soluble test, to be compared ~,vith about 50,000 cells in the RT capture test (Figure 7). An analysis of the inhibiting effects of the cell e~tract on the capture test revealed presence of proteolytic enzymesduring the RT binding step. Incorporation of 2 mM phenyl-methylsulphonyl-fluoride (PMSF) and prA (20 g/ml) in the RT binding step eliminated most of the inhibiting effects of the cell extracts on the capture test. Addition of the same components to the reaction mixture of the soluble test had no effect (Figure 7).As can be seen from Table 4 the RT capture test has a similar capability - of recovering RT from other types of cell extracts. As regards ficol and different types of anticoagulants the table indicates that EDTA is not especially suitablefor testing of blood cells with the RT capture test.
Table 2 shows the linearity with time in the presence of cell extract. From 5 the table, which analyses the effect of a constant amount of HIV-1 RT, it can be seen that the product recovery is linear with time up to 14.5h, provided that extract from no more than 500,000 PBL cellslsample are present. Addition of more cell e~ctract resulted in reducecl product recovery in long term test.

Immunological determination of the reaction product The ~nalysis system according to the invention can be combined with clifferent types of systems for product determination. Figure 8 shows a comparison bet~veen our standard system for detection, ~,vith the use of radiolabelled nucleo-side substrate and a colorimetric system based on Ap conJugated rat antibodies.
As can be seen in Figure 8 the relation was linear between the values in the twosystems. This sho~,vs that the RT capture test easily can be adapted for colori-metric product detection. The detection sensibility in today's colorimetric systems is a function of the incubation time in the RT test step, but also of the time for the final Ap analysis. The Od405 values in Figure 8 were obtained after ~0 minutes of Ap reaction. A stronger signal could be obtained when using a longer i ncubation time.

Capture test for Herpes Simple~ type 1 DNA polymerase Five mouse-~Iabs which were produced against herpes virus type 1 UL42 polymerase complex (Wilcock 1991) were immobilized on plastic beads using the same procedure as the one described above for anti-HIV-1 RT beads. E.~tracts from BHK21-13S cells infected with HSV 1 (strain C42) was produced according to known prior art (Gronowitz and Kallander 1980).
The samples were diluted 1:1 in a buffer containing: 10 mM Tris, pH 7.6;
MgCl2, 4 mM; KCl, 0.15 M; Triton-X 100, 0.5C/c; and NaN3, 0.05 mg/ml. In the first analysis step a set of tubes containing a polystyrene bead with immobilized anti UL42 MAb and 200 f~l sample dilution tube, at 33~C for 1.5h to let MAb bindto the UL42 polymerase complex. The beads were then washed four times with ~ buffer consisting of 3 mIvI Tris, pH 7.6 and O.lC~c of Triton-X 100 for removing disturbing factors coming from the samples. In the ne~ct incubation step the polymerization reaction was started by adding 200 ul of a complete DNA poly-merase reaction mixture per tube to the immobili~ed MAb-UL42 polymerase comple2c. The final concentrations for the non-speed restricted components were as follows: Tris-HCl, 10 mM, pH 7.6; MgCl2, 4 mM; KCl, 200 mM; DTE 10 mM;
Spermidine, 1 mM; Triton-X 100, 0.55~c; GTP, 100,uM; bovine serum albumine, 0.5 5 m~lml. odT22 (1 ~g/ml) was used as primer, pdA300 (10,1~glml) as template and 1.5x10 7 M of 3H-dTTP (specific activity 40-80 ci/mmoles) as nuc}eotide substrate.
The continued treatment of the beads was carried out as in the HIV RT capture test.

CA 0223370l l998-04-Ol Table 1 Remain. Recoverv of incorPorated radioact. RT
RT after RT capture test inhib.
act. test af~er bind Mab beads in free nucleicremain.
to with: acids with: acti-Epi- Mab No No vity tope beadssalt KCl salt ~Cl (Yc) Clone No.* (5'c)(cpm)(cpm) (cpm) (cpm) Column I II III IV V VI VII VIII
Non-HIV
(a-urokinase) - 100705 270 543 110 100 Al(940C6) 1 1031806 501 2480 106 78 Bl(950Cll) 1 109128 126 413 106 69 C2(1.148F2) 2 852665017509 683 973 60 D2(1.149B6) 2 518893 54436 1200 2753 72 E2~1.150E2) 2 523023558691 2160 1303 112 F2(1.158E9) 2 452554635764 2983 2363 60 G2(1.158F7) 2 891492210708 4560 543 76 H2(1.166C2) 2 574466664770 2550 2696 90 J3(1.151F8) 3 1011122 282 1066 196 38 K3(1.152B3) 3 5520827 2759 1073 193 59 L3(1.158E2) 3 883905 497 3636 376 36 M3(1.163C4) 3 883560 2302 2536 476 17 N4(1.153G10) 4 104149 23 153 103 75 05(996E5) 5 94 80 63 1420 220 74 P6(1.009G9) 6 933579 1001 3560 703 129 Q7(1.158ElO) 7 603559137476 1810 1230 89 R7(1.160B3) 7 622844013199 2836 993 61 S7(1.162D10) 7 702646912210 1683 930 70 H2+Q7 2+7 457109799309 1136 1456 101 E2+Q7 2+7 596619375114 2350 2486 109 H2+R7 2+7 566556272965 3256 1630 73 H2+S7 2+7 506365479184 2303 1910 ND
D2+Q7 2+7 574452570799 933 2176 76 40 * According to Orvell et al. 1989.

Table 2 Linearity with the time in the presence of cell e~tract Amount of Amount of Product recovery at indicated Correl. coeff. between RT enzyme PBL extract time, ~ of a control theamo~mtofformed without extract productandtesttime samples sample 2.5h 14.5h 3~.5h Sample Control 2.50 x10-14 2.00 X106 84~c 49~ ND 0.972 0.992 2.50 x10-14 1.00 X106 86 63 ND 0.984 0.992 2.50 x10-14 5.00 x105 100 100 ND 0.995 0.992 2.50 x10~14 2.50 x105 110 97 ND 0.996 0.992 2.50 x10-1~ 1.25 x105 108 105 ND 0.998 0.992 Table 3 Product recovery at indicated amount HIV Corr. coeff.~ between in each sample (~c of cell free control) enzyme and amount of product time7.81 x10-17 5.00 x10-5 8.00 x10-14 sample control 2 108 74 65 0.976 0.991 4 80 70 86 0.991 0.96~
16 57 51 86 0.998 0.910 38 14 17 65 0.994 0.810 * Calculated from seven observations Table 4 RT activity after addition of indicated component to binding buffer, recalculated as ~c of control without additive Component in None Blood cells Blood cells, PMSF, binding buffer prA

RT + PMSF + prA 98 45 RT + EDTA 96 19 40 RT + citrate 68 28 97 RT + heparine 61 55 120 RT + Ficol 112 51 96 Addition of indicated cell material blood cells (EDTA) 100 ND 64 2 x 106 PBL (Ficol) 100 5 82 1 x 106 PBL (Ficol) 100 9 go 1 x 106 Activated PBL* 100 ND 72 * Normal peripheral lympocytes which had been isolated with Ficol gradient centrifugation activated with PHA and used as negative control for HIV isolation.

The ability of the RT capture test to detect RT activity in RT infected cell cultures 1~ blind coded cultures, which had been infected with 16 different HIV
isolates representing a great variety of biological properties ~,vere analyzed after expression of RT and P24-antigen. The analyzed samples were taken out every two days and RT analysis was performed by addition of 100 ~l of crude cell structure suspension to the capture test set, whereas 22.5 ml of supernatant wasused in P24 ELISA. Data as reported in Table 4 shows almost perfect agreement between the results in the two tests. There was a strong correlation bet~,veen the RT activity and the found amounts p24 (r = 0.947, p < 0.01, n = 17) when the analysis was restricted to samples which gave significant values in both tests. All of the samples which were positive in the p24 test were also positive in the RT
capture test, whereas 9 samples which were not significant or gave boarderline values in p24 ELISA, were positive in the capture test. This shows that tests had similar detection sensibility for the investigated isolates. When "the code was broken'' after five days it was found that all HIV 1 isolates ~,vhich had been designated "rapid high" in accordance with their behavour during the isolation of them, already had caused a notible RT activity. Five of the seven HIV isolates which had been designated "slow low", i.e. which on isolation grew slowly and give low titres, showed remarkable RT activity after 6 days of incubation. No activity could be detected in the uninfected control cultures or the two HIV-2 5 infected cultures.

Claims (7)

1. A method for determining the activity of a nucleotide polymerizing enzyme in a sample, characterized by the steps of 1) capturing in an initial capture step the enzyme intended for determination by means of a monoclonal antibody which is immobilized on a solid carrier and is capable of binding said enzyme without detrimentally effecting the enzyme activity, 2) in a subsequent step removing contaminants and disturbing factors, 3) starting nucleotide polymerization by adding a reaction solution containing a primer/template construct and nucleotide substrate, the reaction conditions being selected so as to promote permanent association between the antibody-enzyme- and primer/template constructs, 4) when necessary washing away worked nucleotide substrate, primer/-template and reaction solution from the newly synthesized polymer obtained and 5) determining, in a manner known per se, the amount of nucleotide which has been incorporated with the polymer and, based on this determination, determining the activity of the enzyme.

2. A method according to claim 1, characterized in that the enzyme intended for the determination is DNA polymerase.

3. A method according to claim 2, characterized in that DNA polymerase is reversed transcriptase.

4. A method according to anyone of claims 1-3, characterized in that the removal of contaminents and disturbing factors in step 2) is done by washing of the antibody carrier.

5. A method according to anyone of claims 1-4, characterized in that the reaction solution in step 3) also contains essential salts and co-factors.

6. A method according to anyone of the proceeding claims, characterized in that the determination in step 5) is done by radioactive or non-radioactive marking.

7. Use of the method according to any one of claims 1 and 6 for the determination of HIV1 RT- and Herpes Simples DNA polymerase activity in a sample.