Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.     

Fluorescently imaged particle counting immunoassay for sensitive detection of DNA modifications

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.     

Electrochemical immunoassay of carcinoembryonic antigen based on a lead sulfide nanoparticle label

We describe a lead sulfide nanoparticle (PbS NP)-based electrochemical immunoassay to detect a tumor biomarker, carcinoembryonic antigen (CEA). Cubic PbS NPs were prepared and functionalized with thioglycolic acid (TGA), which stabilized the formed NPs and offered carboxyl groups to conjugate with CEA antibodies. PbS NP conjugated with monoclonal CEA antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary CEA antibody (immobilized on the carboxyl-modified magnetic beads), CEA and the PbS-labeled secondary antibody (PbS-anti-CEA), PbS labels were captured to the magnetic-bead (MB) surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured PbS was used to quantify the concentration of CEA after an acid-dissolution step. The MBs and the magnetic separation platform were used to integrate a facile antibody immobilization with immunoreactions and the isolation of immunocomplexes from reaction solutions in the immunoassay. The voltammetric response is highly linear over the range of 1-50?ng?ml(-1) CEA, and the limit of detection is estimated to be 0.5?ng?ml(-1). The performance of this nanoparticle-based electrochemical immunoassay was successfully evaluated with human serum spiked with CEA, indicating that this convenient and sensitive technique offers great promise for rapid, simple and cost-effective analysis of tumor biomarkers in biological fluids.     

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.     

On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin

By integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device, we have developed a microanalytical platform for performing electrophoresis-based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quantitation of bound and unbound antibody or antigen. To retain biological activity of proteins and maintain intact immune complexes, nondenaturing polyacrylamide gel electrophoresis conditions were investigated. Both direct (noncompetitive) and competitive immunoassay formats are demonstrated in microchips. A direct immunoassay was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off-chip incubation step, the immunoassay was completed in less than 3 min and the sigmoidal dose-response curve spanned an antibody concentration range from 0.17 to 260 nM. The minimum detectable antibody concentration was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing unlabeled and fluorescently labeled tetanus toxin C-fragment compete to bind to a limited fixed concentration of tetanus antibody. The immunoassay technique described in this work shows promise as a component of an integrated microfluidic device amenable to automation and relevant to development of clinical diagnostic devices.     

Minimizing nonspecific protein adsorption in liquid crystal immunoassays by using surfactants

In this paper, we report the role of surfactants in minimizing nonspecific protein adsorption in liquid crystal (LC)-based immunoassays in which LC is used as a readout system. Among all surfactants tested, only nonionic surfactant such as Tween 20 can effectively reduce the nonspecific protein adsorption, while maintaining the selectivity of the LC-based immunoassay. We also show that to minimize nonspecific protein adsorption, Tween 20 can be added directly into the antibody solution to a final concentration of 0.8 mM. After the addition of Tween 20, better correlations between the antibody concentrations and the interference colors of LCs can therefore be obtained. For example, when Cy3 antibiotin was used, black, yellow, red, and green interference colors correspond to a concentration of 5, 25, 50, and 100 μg/mL, respectively. This feature gives LC immunoassay a unique advantage over the fluorescence-based immunoassay.     

Effect of poly(ethylene glycol), tetramethylammonium hydroxide, and other surfactants on enhancing performance in a latex particle immunoassay of C-reactive protein

The influence of a variety and combination of both ionic surfactants and different chain lengths of the polyelectrolyte poly(ethylene glycol) (PEG) on the performance characteristics (with particular reference to signal response) of a homogeneous, latex agglutination immunoassay was investigated. The test analyte was human serum C-reactive protein (CRP), and the antibody reagent consisted of a sheep polyclonal anti-CRP IgG fraction covalently coupled to 50-nm-sized latex including a glycine-capped chloromethylstyrene shell. The amount and rate of immunoagglutination was monitored turbidimetrically after sample addition. It was found that 2.5 mmol/L concentrations of the small cationic surfactant tetramethylammonium hydroxide (TMH), when present alone, substantially increased both reaction rates and sensitivity in the lower clinical ranges of CRP concentration when compared to normally used assay conditions containing PEG and the anionic detergent Gafac. The nonspecific binding (NSB) was also found to be unchanged. Evidence is presented that the TMH enhances the actual antibody-antigen interaction as opposed to the known effects of other surfactants in immunocomplex dissociation or in maintenance of colloidal stability. We suggest that the enhancement seen with TMH could be an alternative to PEG and may provide a new means of further extending detection limits. The utility of this type of immunoassay technology could therefore be increased whenever clinically required.     

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.     

DNA-oligonucleotide encapsulating liposomes as a secondary signal amplification means

A novel liposome-based signal amplification system was developed by encapsulating DNA oligonucleotides within antibody-tagged liposomes and subsequently detecting the oligonucleotide with dye-encapsulating liposomes for double signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein from B. anthracis, was captured by one set of antibodies immobilized in microtiter plate wells and detected using a second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasing the encapsulated fluorescein-tagged DNA 25-mer probe, which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally, the amount of oligonucleotide was detected through the addition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed for a approximately 400x lower LOD than detection of the fluorescein-labeled probe alone. Several aspects were investigated, including the encapsulation of various oligonucleotide concentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluorescein antibody coverage on the liposomes; and immobilized anti-protective antigen antibody concentration. We found that the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA 25-mers were successfully entrapped in the liposome, and minimal leakage was observed over the course of 8 months. When used in the sandwich immunoassay, a limit of detection of 4.1 ng/mL protective antigen was observed with an upper limit of 5000 ng/mL. Due to the endless combination of DNA oligonucleotide sequences, this assay lends itself perfectly for multiplexing on the order of tens to hundreds of analytes.     

Online preconcentration by transient isotachophoresis in linear polymer on a poly(methyl methacrylate) microchip for separation of human serum albumin immunoassay mixtures

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM.     

Determination of benzo[a]pyrene-DNA adducts by solid-matrix phosphorescence.

It has been demonstrated for the first time that linear relationships can be obtained between the solid-matrix phosphorescence (SMP) and the percent modification of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts. The samples of DNA were modified with BPDE at levels of 5.0 x 10(-3), 1.0 x 10(-3), 5.0 x 10(-4), 1.0 x 10(-4), 5.0 x 10(-5), and 1.0 x 10(-5%). In addition, the changes in the SMP intensities of a given percentage of DNA adduct were investigated as a function of sample size, and linear relationships were also acquired. With the different percentages of modified DNA, very good reproducibility of the SMP signals was obtained. Data were acquired with both Whatman 1PS paper and 30% TlNO3/sodium acetate as solid matrixes. The limit of detection for the BPDE-DNA adducts was 2 adducts in 10(7) bases for both Whatman 1PS paper and 30% TlNO3/sodium acetate. In addition, it was shown that it would be important to develop a standard procedure for the preparation of the BPDE-DNA samples if different batches of DNA were used in the preparation procedure.     

Method for identifying nonspecific protein-protein interactions in nanoelectrospray ionization mass spectrometry

The nonspecific self-association of proteins in nanoflow electrospray ionization mass spectrometry (nanoES-MS), and the influence of experimental conditions thereon, are investigated using the protein ubiquitin (Ubq) as a model system. Extents of nonspecific protein association generally increase with protein concentration and, interestingly, with decreasing ES spray potential. The extent of self-association is also sensitive to the duration of the accumulation event in an external rf hexapole. Notably, the relative abundance of metal (Na+ and K+) adducts generally increases with the size of nonspecific Ubq multimer. This result suggests that the gaseous ions of monomeric and nonspecific multimeric Ubq have, on average, different ES droplet histories, with monomer ions originating earlier in the ES process than the nonspecific multimeric complexes. This finding forms the basis for a new method for distinguishing between specific and nonspecific protein complexes in ES-MS. A reporter molecule (Mrep), which does not bind specifically to the proteins and protein complexes of interest, is added to the ES solution at high concentration. The distribution of Mrep bound nonspecifically to gaseous ions of the proteins and protein complexes, as determined from the ES mass spectrum, is used to determine whether a given protein complex originates in solution or whether it forms from nonspecific binding during the ES process. The method is demonstrated in cases where the ions of protein complexes detected by nanoES-MS originate exclusively from nonspecific association, exclusively from specific interactions in solution, or from both specific and nonspecific interactions.     

"Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.     

Interfacial Assembly of Signal Amplified Multienzymes and Biorecognized Antibody into Proteinosome for an Ultrasensitive Immunoassay

Enzyme as signal tag has been widely employed in colorimetric immunoassays for decades. Nevertheless, it remains a great challenge to substantially improve the detection sensitivity of enzyme‐based immunoassays, which inhibits further critical applications. To circumvent this confinement, a multifunctional self‐assembled proteinosome based on the integration of signal amplification elements (enzyme) and biorecognition unit (antibody) is proposed for fabricating an immunoassay strategy with significantly enhanced sensitivity. Owing to the self‐assembly technique, this proteinosome not only efficiently loads abundant enzymes to possess high catalytic activity, but also enhances enzymatic stability and maintains recognition ability of antibody. Using imidacloprid as a model target, the proteinosome‐based immunoassay reaches a limit of detection down to the picogram mL^?1 level, which is 150‐fold lower than that of conventional enzyme‐linked immunosorbent assay. This method provides a versatile approach for constructing spherical proteinosome as a recognizer and amplifier for profiling a broad range of target antigen.     

Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label

A novel, sensitive chemiluminescent (CL) immunoassay has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold label. First, the sandwich-type complex is formed in this protocol by the primary antibody immobilized on the surface of magnetic beads, the antigen in the sample, and the second antibody labeled with colloidal gold. Second, a large number of Au3+ ions from each gold particle anchored on the surface of magnetic beads are released after oxidative gold metal dissolution and then quantitatively determined by a simple and sensitive Au3+-catalyzed luminol CL reaction. Third, this protocol is evaluated for a noncompetitive immunoassay of a human immunoglobulin G, and a concentration as low as 3.1 x 10(-12) M is determined, which is competitive with colloidal gold-based anodic stripping voltammetry (ASV), colorimetric ELISA, or immunoassays based on fluorescent europium chelate labels. The high performance of this protocol is related to the sensitive CL determination of Au3+ ion (detection limit of 2 x 10(-10) M), which is 25 times higher than that by ASV at a single-use carbon-based screen-printed electrode. From the analytical chemistry point of view, this protocol will be quite promising for numerous applications in immunoassay and DNA hybridization.     

A core-shell-type fluorescent nanosphere possessing reactive poly(ethylene glycol) tethered chains on the surface for zeptomole detection of protein in time-resolved fluorometric immunoassay

To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymerization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH(2) nanosphere). The fluorescent NH(2) nanosphere was constructed by incorporating fluorescent europium chelates with beta-diketonate ligands in the core of the NH(2) nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH(2) nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for alpha-fetoprotein (AFP) determination. Anti-human AFP Fab' fragment was covalently conjugated onto the maleimide nanosphere (Fab' nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 x 10(-)(21) mol, M(w,AFP) = 70000) for AFP. The imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when approximately 1.9 x 10(9) particles of the Fab' nanosphere were applied to the microtitration well.     

Nonspecific protein-carbohydrate complexes produced by nanoelectrospray ionization. Factors influencing their formation and stability

Factors influencing the formation of nonspecific protein-carbohydrate complexes during the nanoelectrospray (nanoES) process have been investigated. Protonated and deprotonated nonspecific complexes of ubiquitin (Ubq) and protonated complexes of carbonic anhydrase (CA) with carbohydrates, ranging in size from mono- to tetrasaccharide, were produced by nanoES and detected with a Fourier transform ion cyclotron resonance mass spectrometer. Both the fraction of protein engaged in nonspecific binding with the carbohydrates and the number of carbohydrates bound to the protein increase with increasing carbohydrate concentration. At a given concentration of protein and carbohydrate, nonspecific binding is favored for small (mono- and disaccharide) or hydrophilic carbohydrates over larger or more hydrophobic molecules, which tend to form gaseous monomer or cluster ions by nanoES. However, the extent of nonspecific binding is insensitive to the structure of the protein, with similar distributions of nonspecific complexes observed for both CA and Ubq. Nonspecific association is also insensitive to the charge state of the complex. A comparable degree of binding is observed for complexes in their protonated and deprotonated forms. Furthermore, the number of bound ligands can exceed significantly the charge state of the complex. Thermal dissociation experiments performed on the gaseous nonspecific complexes reveal that their kinetic stability is sensitive to both the structure of the carbohydrate (i.e., mono- < di- < tri- < tetrasaccharide) and the protein (Ubq < CA) and to the charge state, although no simple relationship between stability and charge state was identified. Taken together, the results of this study suggest that neutral protein-carbohydrate interactions (e.g., hydrogen bonds) contribute significantly and, perhaps, predominantly to the formation and stabilization of the nonspecific complexes. A strategy to minimize the formation of the nonspecific complexes, which is based on the enhancement of gaseous carbohydrate ion formation through the addition of metal salts (e.g., CaCl2) to the nanoES solution, is demonstrated.     

An electrochemical metalloimmunoassay based on a colloidal gold label

A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).     

Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes

We report a fully automated sandwich immunoassay for the determination of human insulin using antibody-protein A-bacterial magnetic particle (BMP) complexes and an alkaline phosphatase-conjugated secondary antibody. BMPs bearing protein A-MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp. AMB-1 transconjugant for a protein A-magA fusion gene. MagA protein was used as an anchor to attach protein A onto the membrane. Protein A-BMP complexes harvested from transconjugant AMB-1 were subsequently complexed with anti-human insulin antibodies by specific binding between the Z domain of protein A and the Fc component of IgG to form the antibody-protein A-BMP complexes. The complexes were quite monodisperse after the binding of the antibody. The BMPs' monodispersity resulted in high signal and low noise in the immunoassay. The luminescence intensity ((kilocounts/s)/microg of antibody) from antibody-protein A-BMP complexes after immunoreaction was higher than that from BMPs chemically conjugated to an antibody. This was explained by a difference in dispersion. The fully automated sandwich immunoassay system using antibody-protein A-BMP complexes made possible precise assays of human insulin in serum.