Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC-labeled antibody

Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 microm in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the microchannel. Due to strong non-specific adsorption of microbeads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C. parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.     

Capture and retention of Cryptosporidium parvum oocysts by Pseudomonas aeruginosa biofilms

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

Non-labeled detection of waterborne pathogen Cryptosporidium parvum using a polydiacetylene-based fluorescence chip

A non-labeling fluorescence sensor system was developed using polydiacetylene (PDA) liposomes composed of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a 8:2 molar ratio. The PDA liposomes were immobilized onto an amine-coated glass surface using peptide bonding between the carboxyl group of the liposome and the amine group of the glass surface. The optimum ratio of the cross linker (NHS/EDC) to PDA liposome was determined to be 50% for strong immobilization of the liposomes. Residual carboxyl groups of the PDA liposomes were selectively biotinylated, followed by sequential binding of streptavidin and biotin-antibody (bioreceptor). Finally, the performance of the PDA liposome chip was tested for detecting Cryptosporidium parvum, and yielded a detection limit of 1 x 10(3) oocysts/mL. From these results, it is expected that the PDA liposome chip will have high application potential for the detection of waterborne pathogens including C. parvum.     

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.     

Role of adult sheep in transmission of infection by Cryptosporidium parvum to lambs: confirmation of periparturient rise

In sheep farms, oocyst shedding by asymptomatic adult carriers is one of the mechanisms which may explain maintenance of infections by Cryptosporidium parvum between lambing periods. The objective of this work was to investigate this hypothesis and the existence of a periparturient rise in oocyst shedding. Fourteen pregnant sheep were randomly selected from two farms with a history of neonatal diarrhoea caused by C. parvum and samples were collected from the 6th week before birth until 2 weeks after birth. Faecal samples were filtered, concentrated and examined for oocysts using an indirect immunofluorescence assay. The kinetics of anti-C. parvum antibodies (IgG and IgA) were studied using an indirect enzyme-linked immunosorbent assay. All except one animal excreted C. parrum oocysts at some time during the experimental period. The percentage of animals passing oocysts increased in the first week post-partum (farm 1) and in the first week before birth (farm 2). The numbers of oocysts excreted ranged from 20-440 oocysts g(-1) of faeces. In contrast, no significant changes in the anti-C. parvum immunoglobulin levels were observed over the sampling period. Finally, a high percentage of lambs (71%) born to these ewes acquired infection in the first 2 weeks of life.     

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples.

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

Capture molecules preconditioned for kinetic analysis of high-affinity antigen-antibody complex in Biacore A100

Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.     

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.     

Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies

Cryptosporidium parvum antigens were characterized by immunoblot analysis of sera and intestinal secretions of BALB/c mice orally infected with 10(5) oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by anti-C. parvum IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no cross-reactivity with other C. parvum antigens. Immunofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. parvum and for vaccination.     

The effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum on beef surfaces

AIMS: The aim of this research was to examine the effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum oocysts attached to a beef surface. METHODS AND RESULTS: This study examined the effects of heat treatment (60 or 75 degrees C) on the viability of C. parvum oocysts inoculated onto the surface of beef muscle estimated by vital dye assay. The infectivity of the oocysts was assessed against monolayers of HCT-8 cells. At 60 degrees C viability of the oocysts decreased from 100% at T0 to 64.2% at T60. At 75 degrees C the viability of the oocysts decreased from 100% at T0 to 53.7% at T15 and finally to 11.2% at T60. Oocysts were rendered noninfective against monolayers of HCT-8 cells following treatments of 60 degrees C/45 s and 75 degrees C/20 s. CONCLUSION: The washing of carcasses with hot water and standard thermal treatments is sufficient to kill C. parvum on beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that relatively mild heat, currently used to decontaminate and heat treat beef carcasses and to cook meat products, is capable of inactivating C. parvum.     

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.     

Label-free immunosensor based on gold nanoparticle silver enhancement

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.     

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.     

Design of buffer exchange surfaces and sensor chips for biosensor chip mass spectrometry

The feasibility of buffer exchange in biosensor chip mass spectrometry, along with the construction of base sensor chips and use of alternative chip chemistries, is demonstrated in this work. Beta-2-microglobulin (beta2m) was used as an analyte and captured in the first flow cell (FC1) on the sensor chip surface by an immobilized anti-beta2m antibody. Low pH buffer was then used to elute the captured analyte from the flow cell and route it to a second flow cell (FC2) downstream that served as a cation exchanger that retains the analyte. Following additional washes in FC1, the analyte present in FC2 was either eluted with a higher pH buffer (to demonstrate the possibility of elution into a downstream trypsin flow cell), or it was subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis to verify its presence in FC2. In a separate experiment, a gold-sputtered glass slide (base chip) was activated through a formation of 11-mercaptoundecanoic acid self-assembled monolayer and via reaction with 1,1"-carbonyldiimidazole. The activated chip was placed manually into the biosensor and two surfaces (flow cells) were derivatized with antibodies to beta2m and cystatin C (cysC). To evaluate the chip performance, diluted human urine aliquot was injected over the flow cells. Following the surface plasmon resonance analysis, the chip was MALDI-TOF MS analyzed, yielding signals from beta2m and cysC from their respective flow cells. Artifacts arising from the surface chemistries were not observed in the analysis.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples

Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to 272, average 1,503+/-475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water; however, the method requires further development to improve sensitivity and account for background signals.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.