The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products II. The determination of binding capacity, binding ratio and coefficient of variation of different ELISA plates in sandwich and indirect ELISA

The different commercially available enzyme-linked immunosorbent assay (ELISA) plates were compared for their binding capacity for purified foot-and-mouth disease virus antigen or IgG, their binding ratio (a measure of the efficiency with which positive and negative serum samples may be distinguished), and their coefficients of variation within a plate, between plates and between batches of plates. No one plate could be described as having ideal characteristics, and the choice of ELISA plate depends on the use to which the ELISA is being put. For our purposes, viz. a 'spot-test' which rapidly and efficiently detects specific antibody when the levels of that antibody are low (hybridoma culture supernatants) or when the antibody is contaminated with other 'interfering' proteins (high concentrations of serum), we found that most of the PVC plates and, of the polystyrene plates, the Nunc Immunoplate I and Dynatech M129B plates performed well. The lowest coefficients of variation were obtained using Nunc Immunoplate I, Dynatech M129B and Falcon 3912 plates.     

The potency determination of human varicella-zoster immunoglobulin by enzyme-linked immunosorbent assay, complement-fixation test and indirect fluorescent antibody tests

Traditionally, plasma for the production of the human varicella-zoster immunoglobulin (VZIG) has been selected on the basis of the complement-fixing antibody (CFA) titre. Since immune individuals may lack CFA to varicella-zoster virus (VZV), non-CFA may be of importance in protection. In a search for a simple and reliable method for potency determination, 24 VZIG preparations were quantified by enzyme-linked immunosorbent assay (ELISA), the complement-fixation test (CFT), the indirect fluorescent antibody test to acetone-fixed (IF) and viable (FAMA) VZV-infected cells, respectively. The antibody titres obtained by the various methods were compared. Arranged in order of decreasing agreement, the correlation coefficients (r) of the regression equations between the variables were 0.62 for CFT and FAMA, 0.50 for CFT and ELISA and 0.26 for CFT and IF in a log2 plot. There was complete agreement between the titres obtained by the commercially available Enzygnost Varicella/Zoster kits (Behring Institute, Marburg, F.R. Germany) and the ELISA microtitre plates produced at our institute (r = 1). The regression equation lines for ELISA/CFT and FAMA/CFT titres tended to be parallel to each other, while the line for IF/CFT titres had a less steep slope. Similar titration curves were obtained for VZIGs fractionated by two different methods. Furthermore, the titration curves of serum pools from varicella and zoster convalescents, respectively, had a similar shape below delta OD = 0.4. Generally, a steeper slope was observed above delta OD = 0.4. As antibody detectable by ELISA seems to correlate with protection and the method is sensitive, specific, reproduceable, simple to carry out and easily automated, it may be suitable for the potency determination of VZIGs.     

The use of the method of the complete advisability function for evaluating the quality and standardization of the plates in solid-phase EIA

An attempt to use the method of the complete advisability function for the evaluation of the quality of plates and their standardization in the enzyme-linked immunosorbent assay (ELISA) has been made. Correlations have been empirically established, thus making it possible to calculate the optimum proportions of different ELISA factors: the dose of the antigen (antibody) and the time of sorption, which ensures the best ELISA results with a given type of plates.     

Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus

An enzyme-linked immunosorbent assay (ELISA) for detection of CAR bacillus antibody in rat sera was developed by Ganaway et al., in 1985 although the ELISA method was not described in detail. We investigated antigen preparation and test procedures of the ELISA using two strains of CAR bacillus which we isolated from a mouse (CB-M) and a rat (CB-R). Allantoic fluids containing 2.4 X 10(8)/ml of CB-M and 2.0 X 10(8)/ml of CB-R were washed with sterile phosphate buffered saline (PBS), resuspended in a 1/5 volume of sterile carbonate buffer (pH 9.8) and sonicated. Then 1/40 and 1/80 dilutions of CB-M and CB-R lysates in PBS, respectively, were used for antigen solutions of ELISA. Briefly, antibodies in sera are reacted with antigens coated on the surface of microtiter plates. The amount of horse radish peroxidase labeled protein-A or anti-rat IgG bound to the antigen-antibody complexes is measured on the spectro photometer at wave length of 492 nm. A total of 180 mouse and 205 rat sera were tested against both antigens. The optical density (OD) values of 140 mouse and 161 rat sera obtained from SPF mice and rats free from CAR bacillus infection were on the average 0.005 and 0.019, respectively. On the other hand, OD values of the sera collected from CB-M or CB-R infected animals ranged from 0.20 to 1.52. According to these results, the cut-off OD value for positive reaction was set at 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of ELISA to monitor bacterial kidney disease in naturally spawning chinook salmon

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum (Rs) is a serious problem among Pacific Northwest salmon hatcheries and has raised concerns that salmon reared in hatcheries may spread BKD to natural populations. In order to monitor the potential spread of this disease to salmon spawning in nature, a method must be available to collect and analyze tissues from naturally spawning salmon. Kidney tissue analyzed by enzyme-linked immunosorbent assay (ELISA) is the standard method to detect the presence of Rs in salmon sampled in hatcheries. In this study, we tested the validity of using ELISA on kidney tissue collected from intact carcasses recovered on the spawning grounds to monitor BKD in naturally spawning populations by comparing ELISA optical density (OD) values from kidney tissue that was subjected to conditions that simulated decomposition in a carcass and collection during a spawning ground survey with samples freshly collected from salmon at a hatchery. Mean ELISA OD levels were 1.060 for the samples prepared by the normal preparation and 1.115 for samples prepared by simulating spawning ground survey collection. There was no significant difference in mean ELISA OD between the 2 sample preparations and the relationship did not significantly differ from 1:1 (slope = 0.946). This demonstrates that BKD prevalence in natural populations can be monitored using ELISA conducted on samples from intact carcasses recovered on spawning ground surveys. This will be an important tool for monitoring the effect of hatchery supplementation on naturally spawning salmon populations.     

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV).

An improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) is proposed. The method is based on the use of IgG purified from immune rabbit serum conjugated with biotin. Optimized and validated materials for the test can be stored for a long time in the form of ready-to-use kits. Optimization included selection of anti-poliovirus rabbit antibody batches with the best specificity to D-antigen as well as finding the most efficient parameters for all steps of ELISA protocol. The assay is based on direct ("sandwich") ELISA scheme, in which antigens are captured on ELISA plates coated with purified rabbit polyclonal D-antigen specific IgG raised against wild polioviruses of three serotypes. D-antigen specificity of the IgG was at least 10 times higher than to H-antigen (heat-inactivated virus). The presence of antigen was detected using biotin-conjugated IgG from the same source. Eight-point dose-response curves were obtained for each sample and the reference vaccine. The protocol ensured low background (less than 0.2 OD), linear response over the entire range of optical density measurements (up to 3.0 OD), and high precision of data (assay variability was about 3%). The quantitative results and the validity of the test were determined by two numerical approaches, linear regression and a new analysis procedure called the local interpolation method. For the first approach we also proposed a new method for testing of parallelism of regression lines. The ELISA protocol for all three types of poliovirus is based on standard off-the-shelf reagents, and is highly reproducible and reliable. An in-house Reference Reagent was formulated and calibrated against the International Reference for IPV.     

口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立

抗原纯净度是口蹄疫(Foot-and-mouthdisease,FMD)灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白(Non-structuralprotein,NSP)抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)NSP3A单克隆抗体和辣根过氧化物酶(Horseradish peroxidase,HRP)标记的3B单克隆抗体,建立定量检测NSP3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值(OD)的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0ng/mL之间;而纯化后的...     

一种更灵敏的特异性检测H9亚型禽流感抗体的间接ELISA方法的建立

H9亚型禽流感在全球范围内广泛流行,控制其传播需监测H9亚型禽流感病毒的感染情况及疫苗的免疫效果。为了建立便于检测且灵敏特异的H9亚型禽流感抗体间接ELISA方法,本实验利用不同亚型之间变异较大的H9亚型禽流感病毒HA蛋白的头部球状区作为包被抗原,确定了最佳复合封闭液和抗体稀释液,提高了其特异性。结果显示建立的ELISA方法灵敏度高于血凝抑制试验(HI),且与H3N2、H5N2、H7N9亚型流感病毒及新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、鸡传染性法氏囊病毒(IBDV)和产蛋下降综合征病毒(EDSV)的阳性血清均无交叉反应。另外,利用该方法及HI试验对200份临床鸡血清样本进行检测,两种检测方法的符合率达97%,且存在较高的相关性(R2=0.981 1)。     

Precision determination for the specific oxygen uptake rate (SOUR) method used for biological stability evaluation of compost and biostabilized products

This work represents the first attempt to evaluate the precision of the specific oxygen uptake rate method expressed in terms of repeatability (r) and reproducibility limits (R). Three laboratories were involved in an inter-laboratory test for the validation of respiration analyses on six biomass samples (three composts and three biostabilized products) having different degrees of biological stability. Both the maximum specific oxygen uptake rate peak (SOUR) and the cumulative oxygen demand after 12 h (OD(12)) and 20 h (OD(20)) of respiration test were investigated. Precisions expressed as the relative standard deviation were in the range of 9-41%. Linear regressions found for r and R, versus OD(12) and OD(20), enabled derivation of precision values (r and R) for all respirometric levels within the operating range. The OD(12) and OD(20) indices were found to be more adequate to indicate biological stability since they were less influenced by random errors than the SOUR index.     

Detection of anti-parelaphostrongylus tenuis antibodies in experimentally infected and free-ranging moose (Alces alces)

Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5-9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3-30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61-243 days post-inoculation (DPI). Five animals tested between 16-25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (r = 0.98, P = 0.02 and r = 0.95, P = 0.04, respectively) among animals harboring adult worms (n = 4) but not significantly with the number of worms recovered postmortem (peak OD, r = 0.82, P = 0.18; terminal OD, r = 0.93, P = 0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.     

黄曲霉毒素B_1抗独特型抗体的制备和应用研究II.抗独特型抗体应用

以抗独特型抗体(anti-idiotypeantibody,Ab2)的酶切片段Fab2替代黄曲霉毒素B1(AFB1)建立一种不需要使用AFB1的无毒酶联免疫吸附(Enzyme-LinkedImmunosorbentAssay,ELISA)试剂盒,研究该试剂盒特异性、稳定性和AFB1的加标回收,并将该试剂盒用于农产品和饲料中AFB1的检测。结果表明,该试剂盒具有和常规ELISA试剂盒一样的特异性和加标回收能力,无毒试剂盒和常规试剂盒一样都可以用于农产品和饲料中AFB1的检测,并且两种试剂盒的检测结果并无显著差异,无毒ELISA试剂盒在适当处理后,40C放置3个月和-200C放置5个月,以间接非竞争ELISA测定的吸光值分别是起始时的85%和87%。     

Parasite-specific antibody profile in the aqueous humor of rabbits with ocular toxocariasis

Human ocular toxocariasis is diagnosed using ophthalmologic and immunologic examinations. Many researchers have suggested that intraocular parasite-specific antibody levels are indicative of ocular toxocariasis, but little is known about the time course of the changes in these levels. We therefore investigated the anti-Toxocara canis antibody profile in the aqueous humor in an animal model of ocular toxocariasis. We intravitreally injected T. canis larvae into the right eye of 4 rabbits; 2 rabbits were orally administered T. canis eggs. We collected serum, aqueous humor, and tear samples weekly and determined the serum and aqueous humor levels of anti-T. canis immunoglobulin (Ig)G, IgA, IgM, and IgE antibodies and the tear IgG antibody level by enzyme-linked immunosorbent assay (ELISA). The severity of vitreous opacity and the aqueous humor IgG levels (measured using optical density [OD]) changed concordantly in the larvae-injected eyes; the OD exceeded 0.1 from 2–4 weeks after infection and remained elevated during active intraocular inflammation. However, the aqueous humor IgG levels were also elevated in 6 out of 8 eyes without intraocular larvae in both groups, and were low in 1 eye with live intravitreal larvae. In contrast, the serum IgG and IgM levels and the tear IgG levels increased in all rabbits, regardless of the presence of intraocular inflammation. Vitreous opacity occurred in all intravitreally infected eyes, but significant histopathological evidence of retinal damage was not detected. Thus, besides the presence of intraocular larvae, some other factors in the host may be required for the development of retinal lesions.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B2 (TXB2). As a specific antigen, the bovine serum albumin conjugate of TXB2 was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB2 per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB2 per sample. The suitability of the assay was checked with TXB2-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r = 0.960 and r = 0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r = 0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

Angiopoietin-like protein 3: development, analytical characterization, and clinical testing of a new ELISA

The aim of our work was to develop an assay for the determination of angiopoietin-like protein 3 (Angptl3) in human blood, and investigate its levels in healthy volunteers and donors suffer from metabolic syndrome and familiar hypercholesterolemia. We developed and evaluated the sandwich ELISA method for the quantitative determination of human Angptl3 in serum samples. We conducted also the pilot study on individuals with metabolic syndrome or familiar hypercholesterolemia and healthy probands. The following parameters were measured: blood pressure, waist circumference, Angptl3 serum levels, serum cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, insulin, glucose, A-FABP, and BMI and Quicki insulin sensitivity index was calculated. In the study on 93 healthy volunteers we demonstrated that sex or age is not the determinant for Angptl3 serum values. Futhermore, 118 individuals with metabolic syndrome and 200 patients with familiar hypercholesterolemia were tested and it was found that probands with metabolic syndrome or familiar hypercholesterolemia had higher Angptl3 values than healthy individuals from the first study (medians 289.5 vs. 277.1 vs. 224.8 ng/ml, p < 0.01). All of groups did not differ in sex or age. Angptl3 values correlated with the systolic blood pressure, LDL and A-FABP (p < 0.05). No connection of Angptl3 with triglycerides was found (presumably influences of statins, fibrates via PPARs, etc). However, we performed stepwise regression and found A-FABP and Angptl3 serum values as the independent markers for metabolic syndrome presence only (F ratio 29, p < 0.01). Then we adjusted Angptl3 to A-FABP (reputable metabolic syndrome marker) and recognised that Angptl3 is the A-FABP-independent marker. The pilot study supports the hypothesis about the role of Angptl3 as a new class of lipid metabolism modulator. Their values could be a new key predictors of metabolic syndrome. Further research is necessary to confirm our findings in individuals with dyslipidemia, obesity, CAD and different medication in order to assess Angptl3 value as a risk predictor of accelerated atherosclerosis.     

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.     

Evaluation of Plastic Multi-Well Plates for Serological Screening of Salmonella Cultures with Spicer-Edwards Pooled Antisera

We compared the relative advantages of using glass test tubes and plastic multi-well plates in the serological identification of Salmonella cultures by the Spicer-Edwards method, and we conclude that the advantages of multi-well plates outweigh those of test tubes.     

Immobilization strategies for single-chain antibody microarrays

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.     

酶联免疫吸附试验定量检测血清肝素酶方法的建立及应用

目的：建立一种简便、微创的血清肝素酶酶联免疫吸附（ELISA）定量检测方法,并对肿瘤患者和正常人血清肝素酶进行比较,初步探讨血清肝素酶水平与肿瘤发生、发展的关系。方法：选择鼠抗人肝素酶单克隆抗体和兔抗人肝素酶多克隆抗体,建立双抗夹心ELISA检测方法,并利用此方法检测健康献血者和肿瘤患者血清中的肝素酶水平。结果：组内数据稳定,可重复;肿瘤患者血清中肝素酶D450nm值高于健康献血者。结论：所建立的血清肝素酶ELISA检测方法灵敏、高效,可用于肿瘤的辅助诊断。