Multianalyte microspot immunoassay--microanalytical "compact disk" of the future

Throughout the 1970s, controversy centered both on immunoassay "sensitivity" per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Our theoretical studies revealed that RIA sensitivities could be surpassed only by the use of very high-specificity nonisotopic labels in "noncompetitive" designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodology known as DELFIA--developed in collaboration with LKB/Wallac--represented the first commercial "ultrasensitive" nonisotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of "multianalyte" immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto-unexploited, physicochemical concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is "vanishingly small," fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of "ratiometric" immunoassay, involving measurement of the ratio of signals (e.g., fluorescent signals) emitted by two labeled Abs, the first (a "sensor" Ab) deposited as a microspot on a solid support, the second (a "developing" Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. Our preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 microns 2 or less to be analyzed, and implying that an array of 10(6) Ab-containing microspots, each directed against a different analyte, could, in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte numbers is unlikely ever to be required, the ability to analyze biological fluids for a wide spectrum of analystes is likely to transform immunodiagnostics in the next decade.     

A disposable multianalyte electrochemical immunosensor array for automated simultaneous determination of tumor markers

BACKGROUND: Automated and convenient multianalyte detection with high throughput is increasingly needed in clinical diagnosis. We developed a disposable 4-by-2 array for programmed simultaneous amperometric immunoassay of 4 tumor markers. METHODS: We used a screen-printed technique, 1-step immobilization method, and flow injection technique. We immobilized carcinoembryonic antigen, alpha-fetoprotein, beta-human choriogonadotropin, and carcinoma antigen 125 as model analytes in a redox mediator-grafted, biopolymer-modified, screen-printed carbon electrode array to capture corresponding horseradish peroxidase-labeled antibodies in competitive immunoreactions. The simultaneous multianalyte immunoassay was automatically carried out to amperometrically monitor the mediator-catalyzed enzymatic response to hydrogen peroxide, which decreased in proportion to the concentrations of analytes in samples. RESULTS: The multianalyte immunosensor array had a throughput of 60 samples/h and allowed simultaneous detection of carcinoembryonic antigen, alpha-fetoprotein, beta-human choriogonadotropin, and carcinoma antigen 125 in clinical serum samples with concentrations up to 188 microg/L, 250 microg/L, 266 IU/L, and 334 kIU/L, respectively. The detection limits (limits of the blank, mean of blank plus 3 SD) were 1.1 micro/L, 1.7 microg/L, 1.2 IU/L, and 1.7 kIU/L. The inter- and intraassay imprecision (CVs) of the immunosensor arrays were <7.8% and <9.0%, respectively. The immunosensor arrays were stable for 28 days. CONCLUSIONS: This newly constructed immunosensor array provides a simple, automated, simultaneous multianalyte immunoassay with high throughput, short analytical time, and sufficiently low detection limits for clinical application. This method offers the capability of miniaturizing the multianalyte detection device.     

Dual-label time-resolved immunofluorometric assay of free and total prostate-specific antigen based on recombinant Fab fragments

BACKGROUND: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies. METHODS: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved fluorescence is used to measure both europium and terbium signals in one well. RESULTS: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 microgram/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2-11% and 4-10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0. 99) were not significantly different from zero, and the slopes were 0.95-1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay. CONCLUSIONS: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.     

Gentamicin substrate-labeled fluorescent immunoassay containing monoclonal antibody.

The current substrate-labeled fluorescent immunoassay for gentamicin has been modified by substituting monoclonal antibodies for conventional antiserum to gentamicin. The standard curve generated with gentamicin monoclonal antibody was essentially linear from 0 to 12 micrograms/ml, could detect 0.4 micrograms of gentamicin per ml, and had overall intra- and interassay precision values (coefficients of variation) of 1.9 and 5.0%, respectively. The substrate-labeled fluorescent immunoassay produced with gentamicin monoclonal antibody gave results with clinical specimens comparable (r = 0.991) to those obtained with the commercially available substrate-labeled fluorescent immunoassay and also with an enzyme immunoassay, a radioimmunoassay, and a fluorescent immunoassay. This technologically state-of-the-art assay employs both a nonisotopic label and monoclonal antibodies. It offers excellent precision, sensitivity, lot-to-lot reproducibility, linearity, and reagent stability.     

The Abbott IMx automated benchtop immunochemistry analyzer system

We describe a new clinical laboratory instrument, the IMx, used to automate immunoassay testing in the clinical laboratory. The IMx incorporates a novel technology called Microparticle capture Enzyme ImmunoAssay (MEIA) for assays of high-molecular-mass analytes, and fluorescence polarization immunoassay (FPIA) for hapten assays. A front-surface fluorometer is used to quantify the enzymatic generation of fluorescent product at a rate proportional to the concentration of the analyte in an MEIA, and a fluorescence polarization optical system is used to quantify results in an FPIA. The microprocessor-based instrument uses a robotic arm with two degrees of freedom and a rotating carousel to process the samples for assay. One assay can be done on each of 24 patients' specimens in 30 to 40 min with "walk-away" automation. Calibration curves are stable for at least two weeks. Instrument control involves software-labeled "command keys," a numeric keypad, and an interactive display. Results are output to a thermal printer or computer interface.     

Sample to sample carryover: a source of analytical laboratory error and its relevance to integrated clinical chemistry/immunoassay systems

BACKGROUND: Integrated systems that combine clinical chemistry and immunoassay analyzers are used routinely. Sample to sample carryover is an inherent risk and can cause erroneously high patient test results for immunoassays. IVD manufacturers and laboratories must be aware of this phenomenon and guard against it. METHODS: We used a sample carryover protocol that directs the clinical chemistry module to process samples with very high immunoassay analyte concentrations followed by samples with very low concentrations for the same analyte. Low concentration samples were then tested by the immunoassay module to determine if the clinical chemistry module caused primary sample tube to primary sample tube carryover of the immunoassay analyte. RESULTS: Sample carryover was assessed on the Abbott ci8200 for HBsAg, AFP, beta-hCG, and PSA. Observed HBsAg carryover met the design specification of <0.1 ppm. Carryover for the other analytes was <0.1 ppm or below the assay limit of detection. CONCLUSIONS: IVD manufacturers must design integrated systems to minimize primary specimen tube carryover and avoid analytical laboratory error that can impact patient safety. Carryover testing is difficult for clinical laboratories to perform in order to verify system performance. Laboratories must consider the potential for specimen carryover and its impact on results whether moving primary sample tubes between separate analyzers or using an integrated system.     

A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal anti-testosterone immunoglobulins

An enzyme immunoassay for plasma testosterone was developed based on competition between an immobilised testosterone-casein conjugate and the analyte for polyclonal anti-testosterone immunoglobulins, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised testosterone-casein conjugate was optimised so that the lower affinity anti-testosterone antibody populations present did not affect the assay. The assay standard curve covered a range of 11-300 fmol/well. Testosterone levels in small amounts of male and female plasma could be assayed with good reproducibility and correlated well with results obtained by radioimmunoassay. By comparison with an analogous assay using monoclonal antibodies it appears that, given that the assay sensitivity is the most important criterion for choice, the use a polyclonal antiserum with this type of reactive antibody selection is preferable to the use of monoclonal antibodies.     

Idiometric assay: noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum

We report here a novel noncompetitive immunoassay applicable to the measurement of small-molecular-mass compounds and typified by the direct measurement of estradiol in serum. Two types of anti-idiotypic antibody recognize different epitopes within the hypervariable region of the specific primary antibody (e.g., anti-estradiol). The first anti-idiotype (betatype) recognizes an epitope at the unoccupied binding site, which is masked in the presence of the analyte (e.g., estradiol). The second (alphatype) recognizes an epitope close to the binding site and is unaffected by the presence or absence of the analyte, but is sterically hindered from binding to the primary antibody in the presence of the betatype. The use of these matched antibodies (primary antibody, alphatype, and betatype) has enabled the development of a method for determining antibody occupancy that is not based on a conventional two-site assay. An excess amount of purified monoclonal antibody against estradiol is immobilized onto the walls of microtiter wells. After the capture of analyte, the unoccupied antibody sites are blocked by the addition of an excess amount of betatype. Subsequently, analyte occupancy is determined by the addition of excess europium-labeled alphatype, incubation, washing, and time-resolved fluorometry. The method demonstrates good sensitivity, precision, and comparability with alternative competitive immunoassays.     

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.     

免疫检测干扰因素的分析、识别和对策

免疫检测法中能够改变被检测物浓度或改变被检物与相应检测抗体结合能力的物质,均可能对检测结果造成干扰.内源性干扰因素包括自身抗体、异嗜性抗体、人抗动物抗体及其他一些结合蛋白等.脂血、交叉反应、一些分析前变异、基质效应,甚至检测的设备不同,也可对免疫检测造成干扰.这些干扰可引起许多检测结果的假性升高或降低,包括激素、肿瘤标志、药物、肌钙蛋白和病原微生物的血清学检测等,进而影响临床对疾病的诊断和治疗评估.检验人员与临床医师都应充分认识免疫检测中的干扰因素,并在出现实验室检测结果与临床征象不符时进行充分沟通,查找是否存在干扰因素,并采取适当方案重新检测,以得出正确结果.     

Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes

We describe an amperometric technique for quantification of an enzyme immunoassay in which we use a magnetic working electrode, both to separate bound and free analyte and to monitor the electrochemical response. We used a "two-site" immunometric assay with monoclonal antibodies for human choriogonadotropin (hCG) as a model system in which magnetic particles were used as the solid phase. Separation of bound and free label is readily achieved by localizing the particles at the electrode. Activity of the bound enzyme in the environment of the electrode is determined electrochemically, permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the system is 150 int. units of hCG per litre (1st Int. Ref. Preparation). Correlation between the amperometric measurement of urinary hCG and data for an immunoradiometric assay was r = 0.9. The assay is rapid, requiring a total assay time for each sample of 20 min, which includes 15 min for antibody/antigen binding.     

Multilayer fluorescent immunoassay technique

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.     

Optical biosensor assay (OBA)

We describe a new biosensor immunoassay involving optical diffraction to detect clinically important analytes in human body fluids. A silicon wafer is used as a support for immobilization of antigen or antibody. The protein-coated surface is illuminated through a photo mask to create distinct periodic areas of active and inactive protein. When the surface is incubated with a positive sample, antigen-antibody binding occurs only on the active areas. Upon illumination with a light source such as a laser, the resulting biological diffraction grating diffracts the light. A negative sample does not result in diffraction because no antigen-antibody binding occurs to create the diffraction grating. The presence or absence of a diffraction signal differentiates between positive and negative samples, and the intensity of the signal provides a quantitative measure of the analyte concentration. The technique is demonstrated with a quantitative assay of choriogonadotropin in serum.     

An immunoassay of human band 5 ("tartrate-resistant") acid phosphatase that involves the use of anti-porcine uteroferrin antibodies

We describe an immunoassay for human band-5 acid phosphatase in which antibodies to porcine uteroferrin, immobilized on Sepharose particles, are used. Band-5 acid phosphatase is the tartrate-resistant isoenzyme normally expressed in tissue macrophages such as osteoclasts and alveolar macrophages. The immunoassay is similar in reproducibility and sensitivity to assays based on inhibition by d-tartrate. However, compared with the latter, the greater specificity of the immunoassay makes it markedly less susceptible to errors arising from the presence of non-band-5 acid phosphatases, e.g., from prostate.     

A comparison of four commercial test kits for detection of cytomegalovirus antibodies in blood donors

Four commercial test kits for detecting cytomegalovirus (CMV) antibodies (indirect hemagglutination assay, indirect fluorescent antibody technique, enzyme immunoassay, and passive latex agglutination technique) were compared according to their technical demand, hands-on time, turnaround time, concordance with other techniques, reagent cost per test, and objectivity. The indirect hemagglutination assay, the enzyme immunoassay, and the passive latex agglutination technique produced identical results in 85 donors, detecting 63 positive and 22 negative samples. The indirect fluorescent antibody technique showed discrepant results in four samples. The passive latex agglutination technique rated best overall since it was technically the easiest and required the least hands-on and turnaround times; the short turnaround time (10 minutes) rendered the latex technique a more flexible test for blood bank use because both scheduled and emergency CMV screening of donors could be accommodated. The comparatively high reagent cost of the latex test kit could be offset by savings on technologist time.     

Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime

BACKGROUND: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015-30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44:2036-43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. METHODS: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 microL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. RESULTS: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, < 2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. CONCLUSIONS: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the "ambient analyte" regime because depletion of analyte molecules from the assay volume is insignificant.     

Enzyme immunoassay system for panel testing

An immunoassay system based on enzyme immunoassay technology has been developed for quantitative panel testing. The system includes test card disposables, reagents, and an instrument. Patients' samples are processed semiautomatically in the instrument with minimum user intervention. The test card has multiple test areas at individual locations on a membrane solid phase so that simultaneous determinations from a single specimen are possible. Each panel also includes positive and negative reagent procedural controls. Factory-determined calibration curves for each analyte are provided in barcode form with each test kit. The reagents include a specimen dilution buffer, enzyme conjugate, and precipitogenic substrate. Up to 10 test cards at a time can be processed in random-access and continuous-access modes, with automated agitation of sample and reagents over the solid phase, temperature-controlled incubation, and membrane washing and reading, data reduction, and printout of results. The optical reader measures diffuse reflectance and features source intensity and wavelength compensation.     

Antibody microarrays: the crucial impact of mass transport on assay kinetics and sensitivity

Although they are superficially similar to DNA microarrays, immunoassay microarrays represent a daunting technological challenge owing to the much wider diversity of proteins. Yet, as the leading edge of bioscience migrates from genomics to proteomics, the complexity and enormous dynamic range of proteins in a cell necessitate an analytic tool with exceptional specificity and sensitivity. In theory, microspot immunoassays could fulfill this need. However, antibody microarrays have had limited success to date, and have often required a highly sensitive detection system and/or sophisticated immobilization approach to be of any use for the profiling of complex specimens. There is a solid body of work on the theory of microspot reaction kinetics, yet much of the published experimental work on protein microarray development pays insufficient attention to the kinetic aspects of this interaction. This review explains that one of the main limitations for the sensitivity of current generation microspot immunoassays is the strong dependence of antibody microspot kinetics upon mass flux to the spot. This not only involves migration of analyte in solution, but also across the surface of the solid phase. Understanding of this effect will be discussed, along with several related effects and their significance to improving existing microarray designs. It is concluded that current efforts may be too focused on areas that cannot improve performance significantly, and that other critical areas of design should receive more attention. Finally, the review addresses the question of whether ambient analyte immunoassay is truly a separate category of microspot assay, with the conclusion that this may be a flawed concept.     

Enzyme immunoassay for antibodies to Epstein-Barr virus nuclear antigen.

An enzyme immunoassay that detects IgM and IgG antibodies to the nuclear antigen of Epstein-Barr virus (EBV) is evaluated. The EIA was compared with a rapid monoscreen (Immunoscan-IM) and an indirect fluorescent antibody procedure to determine the correlation of the new test with the current methodology. Among 73 patients tested for heterophilic antibody, 36 were positive; 25 of these had IgM antibodies to nuclear antigen. The sensitivity of the EIA compared to the mono screen was 69.4% and specificity was 97.3%. In comparison with fluorescence assays for IgG to the nuclear antigen, 94 patients were tested with 84 showing positivity by the fluorescent method. Among these, 80 were positive by EIA yielding a sensitivity and specificity of 95.2% and 90.0%, respectively. The EIA does not appear sensitive enough for replacement of the monoscreen but was a sensitive, cost-effective alternative to the indirect fluorescent antibody test.     

Immobilization of antibodies on ultraflat polystyrene surfaces

BACKGROUND: Functional antibody surfaces were prepared on ultraflat polystyrene surfaces by physical adsorption, and the uniform distribution of monoclonal antibodies against hepatitis B surface antigen (anti-HBs) on such surfaces and the presence of dense hepatitis B surface antigen (HBsAg) particles captured by immobilized antibodies were identified. METHODS: A model polystyrene film was spin-coated directly onto a silicon wafer surface. Atomic force microscopy was used to directly monitor the immobilization of anti-HBs antibodies and their specific molecular interaction with HBsAg. Enzyme immunoassay was also used to characterize functional antibody surfaces. RESULTS: A mean roughness of 2 A for areas of 25 microm(2) was produced. We found a uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after incubation with HBsAg. CONCLUSIONS: This study confirmed the potential of preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on polystyrene surfaces with controlled roughness. It is expected that such biofunctional surfaces could be of interest for the development of new solid-phase immunoassay techniques and biosensor techniques.