Monoclonal antibodies to native HIV type 1 reverse transcriptase and their interaction with enzymes from different subtypes.

Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.

Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity.

The development of a colorimetric capture assay for HIV-1 reverse transcriptase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all performed in the same microtitre plate. Mouse monoclonal anti-RT antibodies of subclass G2a were bound by polyclonal goat anti-(mouse IgG2a) immobilized in the wells of a microtitre plate. The monoclonal antibodies (mAbs) were selected for their ability to bind HIV-1 RT without hampering the polymerase activity. The assay system first involved the RT's adherence to the immobilized mAbs. Non-specific enzymes and other impurities were removed by a simple wash, after which an RT reaction mixture containing BrdUTP as nucleotide substrate was added. After the RT reaction substrate and product had been separated by washing of the plate, the amount of BrdUMP-DNA in the wells was finally detected with alkaline-phosphatase-conjugated mouse anti-BrdU antibodies of subclass IgG1. The background signal in this system was similar to the signals obtained with control wells coated with BSA only. A detection limit of 1.2 micro-units of RT activity, corresponding to 0.3 pg of RT protein, was obtained for the capture assay when applying colorimetric product detection. The assay detected RTs from HIV-1 subtypes A and B and one of the two D type isolates tested. None of the five non-HIV-1 RTs tested was found positive. At least 50 microl of human serum or plasma per sample could be included in the capture assay without adverse effects on the recovery of the RT activity.

A colorimetric reverse transcriptase assay optimized for Moloney murine leukemia virus, and its use for characterization of reverse transcriptases of unknown identity.

A non-radioactive reverse transcriptase (RT) assay, reported as useful for lentivirus RTs, was optimized for the measurement of Moloney murine leukemia virus (MMuLV) RT. The optimized assay could detect 0.3 microU of MMuLV RT. The specificities of the MMuLV and lenti RT assays were demonstrated using the RTs of human immunodeficiency virus type 1, simian immunodeficiency virus, feline immunodeficiency virus (FIV), visna virus, human T-cell lymphotropic virus type 1, MMuLV and feline leukemia virus (FeLV). An RT activity blocking antibody (RTb-ab) assay was standardized for Mn2+ dependent MuLV-related RTs. The assay was used to demonstrate the distinct antigenic properties of RTs from mammalian MuLV-related retroviruses and lentiviruses. Cross-reactivity between MMuLV RTb-ab and FeLV RT but not between MMuLV RTb-ab and e.g. FIV RT was demonstrated. An RT activity found in the murine myeloma cell line SP2/0 was found to have similar assay preferences as MMuLV RT, and the MMuLV-RT hyperimmune sera reacted strongly against this RT, indicating the RT to be of MuLV-related etiology. The use of the RT and RTb-ab assays for detection and characterization of RTs of known or unknown identity is discussed.

Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains.

Failure to detect infection with HIV-1 non-B subtypes in some antibody screening assays has been shown. To date, however, no studies have been published evaluating the capacity of standard tests to quantify replication of divergent HIV-1 in cell culture. Reverse transcriptase (RT) activity and p24 antigen assays are the two methods most commonly used for this purpose. A homogeneous panel of HIV-1 subtype B viruses from northern Italy and a heterogeneous panel of diverse genetic subtypes (A to F and O) from different regions of the world were cultured under identical conditions. A new nonradioactive RT assay was used as a basis for comparison to evaluate the capacity of two p24 assays to quantify viral growth in both panels. Comparison of the p24 amount/RT activity (p24/RT) ratios showed that ratios in the subtype B panel tended to be markedly higher than in the diverse subtype panel. Greatest variation was seen with one of the subtype O isolates, where up to a 400 times lower ratio was obtained compared with the average ratio for the subtype B panel. In addition, one Thai subtype B virus also gave a markedly reduced ratio. Furthermore, comparison between the two p24 assays showed different abilities to detect p24 from different HIV-1 isolates. We discuss limitations for the use of anti-HIV-1 p24 antibodies produced by immunization with subtype B p24 in p24 assays.

Colorimetric assays for evaluation of the mode of action of human immunodeficiency virus type 1 non-nucleoside reverse transcriptase inhibitors.

Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-CI-TIBO [(+)-S-4,5,6,7-tetrahydro-9- chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1-jk)(1,4)- benzodiazepin-2(1H)-thione)], nevirapine (6,11-dihydro-11-cyclopropyl-4-methyl-dipyrido[2,3-b:2',3'-e]-[1,4]di azepin- 6-one), MSA-300 (N-[cis-2-(2-hydroxy-3-acetyl-6-methoxy-phenyl)-cyclopropyl]-N'- (5-chloropyrid-2-yl)-thiourea) and delavirdine ¿1-(5-methanesulphonamido-1H-indol-2-yl-carbonyl)-4-[3- (1-methylethylamino)pyridinyl]piperazine¿ were analysed for the mode of action of their inhibition of human immunodeficiency virus type 1 (HIV-1) RT in three different assays utilizing a 96-well microtitre plate format, with solid-phase conjugated poly(rA) as template. These were: (i) direct RT assay, for determination of IC50 values of RT inhibitors; (ii) RT template/primer binding inhibition (BIC) assay, for measuring the effect of various substances on the RT activity binding to template/primer; (iii) RT protein ELISA, for measuring RT protein binding to template/primer with a monoclonal antibody reactive against a peptide in the RNase H region. MSA-300 and delavirdine gave the lowest IC50 values, ranging from 0.17 microM to 0.24 microM for MSA-300 and from 0.12 microM to 0.38 microM for delavirdine, whereas higher IC50 values of approximately 20 microM were obtained for 9-CI-TIBO at all primer concentrations. None of the non-nucleoside substances had inhibiting effects on the binding of template, primer, or template/primer to RT protein. Their inhibition of RT activity was not due to prevention of RT binding to template/primer. TIBO, nevirapine and delavirdine bound to RT reversibly, and they bound more tightly to RT template/primer ternary than to RT template binary complex. MSA-300 showed a comparatively high affinity for the enzyme. The utility of the three assays in relation to screening and analysis of RT inhibitory substances is discussed.

Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay.

Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms of primary infection. In addition, HIV-1 RTb-Ab was detected in the same recently infected individuals in most cases within 1 month and in some cases as early as 10 to 12 days after the onset of symptoms. A cross-reactivity study involving HIV-1 and HIV-2 RTb-Abs and their homologous RT showed HIV-1 RTb-Ab to be highly type specific. None of 10 serum samples from HIV-1-infected individuals showed cross-reacting RTb-Ab toward HIV-2 RT, whereas 4 of 10 serum samples from HIV-2-infected patients showed cross-reactivity toward HIV-1 RT; however, the cross-reactivity toward HIV-1 RT was 3,000 times lower than that toward its homologous RT. Future uses for the assay with reference to the recent World Health Organization proposal for other methods instead of Western blotting (immunoblotting) for confirming HIV-1 infection and for methods for the diagnosis of infection as follow-up in vaccine trials are also discussed.

A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation.

A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine-leukaemia-virus RT. The assay was found to be useful with various types of cell-culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications.

A method for combined immunoaffinity purification and assay of HIV-1 reverse transcriptase activity useful for crude samples.

Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence to the immobilized Mabs. Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added. Substrate and product were finally separated by a wash of the beads. Practically all radioactivity incorporated into DNA (>98%) was recovered on the bead. The Michaelis-Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 x 10(7) PBL cells/ ml. A strong correlation (r = 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.

HIV-1 reverse transcriptase inhibiting antibody titer in serum: relation to disease progression and to core-antibody levels.

A new assay for detecting inhibition of reverse transcriptase activity (the RT-i REA) was developed. This assay was standardized for screening serum samples for reverse transcriptase inhibiting antibodies (RT-iAb). High specificity (100%) and sensitivity (greater than 98%) were achieved with samples from HIV-negative individuals and HIV-infected individuals. The RT-i REA was also used in a study of the titers of RT-iAb in serum samples obtained from 33 HIV-infected homosexual men. The results confirmed the relation between decreasing RT-iAb levels and progression to late stages of the disease. Furthermore, a falling RT-iAb titer was observed in 14 of 15 individuals experiencing periods of severe clinical symptoms attributed to HIV-activity. In 7 of the patients the decline in RT-iAb titer began prior to severe clinical symptoms. The fall in RT-iAb titer also correlated with a reduction in core Ab level. The core Ab level has previously been reported to be a disease progression marker with considerable prognostic value. However, whereas all patients were positive for RT-iAb, 8 of the 33 patients did not have detectable core Ab. The use of RT-iAb titer as a marker of disease progression is discussed.

HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables.

A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.

Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV.

Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies.

Clinical and serologic markers of stage and prognosis in small cell lung cancer. A multivariate analysis.

The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer (SCLC): lactic dehydrogenase (LDH), serum thymidine kinase (S-TK), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and tissue polypeptide antigen (TPA). More traditional clinical and serologic markers were also evaluated. Univariate analysis showed that all of the biochemical markers mentioned above, the Karnofsky index (KI) and the patient's sex were related to both the stage of disease (limited/extensive disease: LD/ED) and to survival. The strongest marker for the clinical stage was S-TK, whereas TPA showed the strongest relationship with survival. Multivariate analyses produced a model consisting of S-TK, CEA, NSE, and the patient's sex for determining the clinical stage. To compare the prognostic capacity of easily determined biochemical and simple clinical variables to the more resource-demanding variable of the clinical stage, three multivariate analyses in relation to survival were performed: (1) biochemical markers and simple clinical variables; (2) LD/ED and simple clinical variables; and (3) all available variables. The model obtained from the first analysis included TPA, KI, age, and the patient's sex; the model from the second analyses included LD/ED, patient's age, and KI; and the model from the third analysis, TPA, KI, age, sex, and LD/ED. Indices based on these three multivariate models were calculated for each patient and the prognostic capacity of these indices was compared. Pretreatment serum marker levels also had the capacity to predict both the grade and the duration of the response to therapy.

Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses.

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.

Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA.

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalovirus (CMV) infection, B12-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (greater than 1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation. The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity. The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.

Antibodies against varicella-zoster virus and herpes simplex virus deoxythymidine kinase in heterologous infections.

Antibody responses to varicella-zoster virus (VZV) deoxythymidine kinase (dTK) and herpes simplex virus (HSV) dTK in homologous and heterologous infections were studied. Antibodies blocking the enzymatic activity of VZV-dTK appeared late after varicella and decreased more or less in parallel with the decreasing complement fixing [CF] titre. In herpes zoster, on the other hand, antibodies to VZV-dTK appeared soon after infection. Antibodies against HSV dTKs appeared long after primary infection, but they were subsequently present in all other HSV-CF positive sera. In recurrent HSV, all acute sera were already HSV-dTK antibody positive, and three of nine persons showed an increase in titer between their acute and convalescent sera. Blocking antibodies to VZV-dTK appeared rapidly in specimens from three of 18 individuals positive by an immunofluorescence VZV-immunity test during HSV infection, whereas all other specimens remained devoid of blocking antibodies against VZV-dTK. A rise in antibody titre against HSV-dTK during VZV infections was observed in serum specimens from three of 13 HSV-CF positive patients, whereas an antibody response against HSV-dTK was not found in HSV-CF negative individuals in connection with VZV infections. The relevance of the sporadic increase in the titres of antibodies against heterologous viral dTKs is discussed.

Detection and characteristics of DNA polymerase activity in serum from patients with malignant, viral, or B12-deficiency disease.

DNA polymerase activity was demonstrated in sera from patients with diseases affecting DNA metabolism in different ways, i.e. malignant, viral and vitamin B12-deficiency disease. Using the current procedure, such activity was only detected in sera with pathological levels of thymidine kinase, i.e. no reference level of DNA polymerase activity in healthy individuals could be established. The activity detected for all three types of disease was similar to that of proliferation-associated DNA polymerase alpha, both with respect to sensitivity to different chemical inhibitors and to inhibition by monoclonal antibody. The levels of activity of DNA polymerase and thymidine kinase showed a wide variation and were not significantly correlated when all DNA polymerase-positive sera were included in the analysis. The variation in the ratio of polymerase to kinase activity within a given disease was smaller and the distributions of the enzyme ratios induced by the three types of disease differed significantly. Considering that DNA polymerase activity can be quantitated directly in crude sera, and that such analyses seems to give biological and clinical information, the development of an assay with improved sensitivity for extensive studies is justified.

Varicella zoster virus deoxythymidine kinase is present in serum before the onset of varicella.

A sensitive enzyme assay with 125I-iododeoxyuridine as substrate and cytidine triphosphate as phosphate donor was used for the direct detection of varicella zoster virus (VZV) deoxythymidine kinase (TK) in human serum. Sera sampled during the incubation period of varicella from 2 patients, a 42-year-old man and his 11-year-old son, have been analysed for TK activity. A simultaneous increase in cellular and VZV TK activity, starting 5 to 3 days before the onset of clinical varicella, was observed.

Biochemical markers in multiple myeloma: a multivariate analysis.

The analysis of individual biochemical and clinical variables in 121 patients with multiple myeloma showed that serum beta 2-microglobulin (S-beta 2m) had the most significant relation to survival. Other variables such as serum thymidine kinase (S-TK), serum lactate dehydrogenase (S-LDH), S-creatinine, haemoglobin (Hb), ESR, S-albumin, age and clinical stage were also significant. No such relationship was found with M-component, presence of light chains in urine, type of secreted immunoglobulin or S-calcium. The exclusion of clinical stage in the first multivariate analysis resulted in a model consisting of S-beta 2m, age and S-TK, none of the other variables gave additional information. When in the second multivariate analysis the basic variables involved in staging procedure were excluded and clinical stage included, stage III, but not stage II, was found to give additional information to the model described above. Individual analysis of the variables showed that Hb had the most significant relation to effect of initial therapy. Other significant variables were S-TK, S-beta 2m and age. When using the multivariate approach, Hb alone was found to contain all the relevant information.

Serum deoxythymidine kinase correlates with peripheral lymphocyte thymidine uptake in chronic lymphocytic leukemia.

The serum thymidine kinase (S-TK) and proliferative activity of the leukemic cells were determined in 27 untreated patients with chronic lymphocytic leukemia (CLL). A significant positive correlation between S-TK and proliferation expressed as a proliferative index (PI) was found (r = 0.70, p less than 0.001). Additionally, PI (r = 0.59, p less than 0.01) and S-TK (r = 0.47, p less than 0.05) correlated to peripheral blood lymphocyte count. When different variables and combinations of variables were studied in order to define their capacity for discriminating between progressive and indolent CLL, S-TK activity and PI proved to be powerful indications. In longitudinal studies, both S-TK and PI paralleled disease activity. A model where a combination of S-TK and PI gives information of the degree of localized disease is proposed.