Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

Background

The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR.

Results

A liposome detection reagent was prepared, which consisted of a population of liposomes ~120?nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose?Cresponse curve from 10^-10?M to 10^-16?M CEA. Within this range the assay coefficient of variance was <6?% for repeatability and <2?% for reproducibility. The assay detection limit was 13?fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed.

Conclusions

The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

The use of avidin-biotin interaction in immunoenzymatic techniques.

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.     

Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.     

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.     

R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.     

Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera

Chemiluminescence has been considered an alternative to radioisotopic detection of materials in the life sciences. One application of this nonradioisotopic method is the electrophoretic mobility shift assay. The essential requirement for quantitative applications is that the chemiluminescence signal is linearly proportional to the concentration. However, the generation of chemiluminescence is a multi-step process, therefore linearity cannot be assumed. Therefore, it is important to verify linearity before applying the method as a quantitative tool. We used a commercial chemiluminescence generating system to evaluate the validity of quantitative measurements of biotin-labeled tRNA and single-stranded DNA. The results indicate that the relationship between the chemiluminescence signals and the quantity of biotin-labeled nucleic acids is hyperbolic rather than linear. However, it was found that with less than 50 fmol of biotin-labeled nucleic acid, which corresponds to 2.5 nM in 20 microl, linearity can be demonstrated within 5% error. Therefore, chemiluminescence-based quantitative measurements are a reliable method within these limitations.     

Comparison of the sensitivity of the indirect,antibody-conjugated and the triple-bridge immunoperoxidase methods for immunohistochemical detection of carcinoembryonic antigen

Summary A comparison between two immunoperoxidase staining procedures, a triple-bridge and an indirect, antibody-conjugated method, was made to determine the relative sensitivity of each technique in the detection of carcinoembryonic antigen (CEA) in routine tissue sections. The enzyme-antibody conjugates for the indirect procedure were prepared according to the method of Nakane and Kawaoi (1974). The indirect, antibody-conjugated method, proved to be slightly more sensitive than the triple-bridge by two criteria. First, CEA could be localized using higher dilutions of the primary antiserum by the indirect technique, and secondly, tissues were shown to stain for CEA in specimens with lower tissue CEA levels by the indirect procedure than by the triple-bridge method. The preparation of enzyme-antibody conjugates is a relatively simple procedure and, in addition, the conjugates will remain stable when kept frozen or at 4°C. Background staining due to non-specific interaction of the conjugate with the tissue can be eliminated easily by incubation with normal serum. These results indicate that the indirect, antibody-conjugated method can be used to enhance the staining of CEA in routine tissue sections.Supported in part by NIH contract NCI-NO1-CB-84257     

Sister chromatid differentiation and chromosomal in situ suppression hybridization: a combined methodology for analyzing cell proliferation and SCEs in individual chromosomes.

A technique combining sister chromatid differentiation (SCD) with chromosomal in situ suppression (CISS) hybridization is described. This combined methodology allows simultaneous analysis of cell-proliferation kinetics and sister chromatid exchanges (SCEs) in chromosomes identified by probes. To demonstrate the usefulness of this approach, cultured fibroblasts from a patient with Pallister-Killian syndrome, mos46/47,+i(12p), a chromosome mosaicism disorder, were studied. The fibroblasts were cultured in the presence of 5-bromodeoxyuridine (BrdU) for 72 h. Chromosome preparations were stained by a modified fluorescence-plus-Giemsa method to obtain SCD. For identification of the normal chromosome 12 and the i(12p), CISS hybridization with a biotin-labeled chromosome 12-specific library probe (LA 12NS01) was carried out after SCD. The hybridization was detected by an indirect immunofluorescence technique. For the analysis of cell kinetics and SCEs, the technique allows rapid, reliable identification of abnormal and normal cell populations. It also allows analysis of SCEs in individual chromosomes.     

Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays

We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein-RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein-nucleic acid complexes. After separation of free from complexed RNA in agarose, the RNA is electroblotted to positively charged nylon. The biotin-labeled RNA is readily bound by a streptavidin-alkaline phosphatase conjugate, allowing for very sensitive chemiluminescent detection ( approximately 0.1-1.0 fmol limit). Using our system, we were able to purify both known iron-responsive proteins (IRPs) from rat liver and assess their binding affinity to RNA containing the iron-responsive element (IRE) using the same batch of biotinylated RNA. We show data indicating that agarose is especially useful for cases when large complexes are formed, although smaller complexes are even better resolved.     

Carcinoembryonic antigen in cytological specimens of urothelial carcinoma

A peroxidase-antiperoxidase (PAP) technique was developed for the detection of carcinoembryonic antigen (CEA) in urothelial transitional cells of 52 bladder cancer patients. The percentage of CEA-containing malignant cells varied from 10% to 100%. As a mean, 65% of the malignant cells stained for CEA, while the corresponding figure for benign-looking cells was 24%. The results were compared with cytological evaluations, flow cytophotometric results, and immunofluorescent (IF) staining for CEA. With increasing malignancy, more CEA was detected with the PAP technique, whereas the IF technique failed to show this trend. 18 of 20 malignant-tumors had an aneuploid DNA pattern. The two diploid cases were moderately well differentiated. Samples from bladders with heavy inflammation should be avoided in the PAP technique, since the unspecific staining of granulocytes disturbed a correct evaluation of the transitional cells. The PAP technique used on cytological material is recommended for antigen determinations, since good morphology is obtained.     

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

Summary A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

Application of the avidin-biotin system for post-embedding cytochemical demonstration of a biotin-labeled IgG tracer

The capacity of the avidin-biotin system for post-embedding cytochemical detection of a biotin-labeled proteinaceous tracer was investigated. By testing modifications of the fixatives, the epoxy embedding medium, and various etching solutions, a procedure was developed to localize specifically the biotinylated tracer on 1-micron thick and thin sections. By use of this technique, a systemically administered IgG tracer was demonstrated after 24 hr throughout the endomysium of mouse skeletal muscle. In the adjoining sciatic nerve, the tracer IgG occurred at the perineurium and within endoneurial blood vessels, although the endoneurium itself was spared because of the presence of the blood-nerve barrier. Because of the small size of the biotin ligand and the non-denaturing method of labeling proteins, our mode of application of the avidin-biotin system appears suitable for tracer studies.     

A novel bioassay for the monitoring of carcinoembryonic antigen in human biofluid using polymeric interface and immunosensing method

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins. Recognition of CEA is needed to monitor the physiological status of the patient for treatment and also it is important to assess the severity of the disease. In this work, we reported a novel sandwich‐type electrochemical immunosensor based on gold nanoparticles functionalized cysteamine‐glutaraldehyde (AuNPs‐CysA‐GA) and it successfully designed to detection of the CEA biomarker in a human plasma sample. The AuNPs‐CysA‐GA provides a large surface area for the effective immobilization of CEA antibody, as well as it ascertains the bioactivity and stability of immobilized CEA antigens. Biotinylated‐anti‐CEA antibody (Ab1) was immobilized on the surface of glassy carbon electrode (GCE) modified AuNPs‐CysA‐GA. Also, secondary antibody (HRP‐Ab2) was costed immobilized to complete the sandwich part of immunosensor. Field emission scanning electron microscope (FE‐SEM and EDS), was employed to monitor the sensor fabrication procedure. The immunosensor was used for the detection of CEA using differential pulse voltammetry (DPVs) technique. The proposed interface led to enhancement of accessible surface area for immobilizing high amount of anti‐CEA antibody, increasing electrical conductivity, boosting stability, and biocompatibility. Finally, the low limit of quantitation (LLOQ) of the proposed immunosensor was obtained as 7 ng/mL with the linear range of 0.001‐5 μg/L. The proposed immunoassay was successfully applied for the monitoring of the CEA in unprocessed human plasma samples. Obtained results paved that the proposed bioassay can be used as a novel bioassay for the clinical diagnosis of cancer based on CEA monitoring.     

Immunohistochemical study of carcinoembryonic antigen (CEA) in normal and embryonic human tissues using a CEA-specific oncoprecipitin, krustacin

Using immunoperoxidase technique, antibodies to CEA (Ab) were compared to a glycoprotein krustacin (Kr) extracted from Pagurus prideauxii, which has an ability to precipitate specifically CEA. It was found that Kr and Ab reacted in a similar manner with embryonic and normal gastrointestinal tissues, revealing practically identical localization of the antigen in the tissues and cells. It was possible, however, to note some quantitative and qualitative differences in the distribution of antigen, which showed that Kr and Ab reacted with different determinants of the CEA molecule.     

Microwave irradiation-stimulated in situ hybridization procedure with biotinylated DNA probe.

A microwave-stimulated in situ hybridization technique using biotin-labeled DNA probe is described. Both hybridization reaction and the detection of the biotin label (with a alkaline phosphatase or immunofluorescence method) has been performed in the microwave oven. All procedures are completed within one hour. The described method was applied for identification of nucleic acid sequences of human immunodeficiency virus in human cell lines. The resolution and the intensity of the signal are as good as from a standard technique with overnight incubation of the probe. Because of the simplicity and speed of the technique, this procedure can be used in a number of other applications.     

Identification and characterization of collagen-binding activity in Streptococcus mutans wall-associated protein: a possible implication in dental root caries and endocarditis

Streptococcus mutans is implicated in coronal and dental root decay, and in endocarditis. Comparative study of the amino acid sequence of S. mutans 47 kDa wall-associated protein A (WapA) revealed a collagen-binding domain (CBD) at the N-terminal region. Recombinant AgA (WapA truncated at the carboxyterminal end) was isolated, biotin-labeled, and analyzed by Solid Phase Binding Assay. The results showed that biotin-labeled AgA bound significantly and in a dose-dependent manner to immobilized collagen type I, and to a lesser extent to fibronectin, but not to collagen type IV or laminin. Binding of biotin-labeled S. mutans cells to collagen-coated surfaces was significantly inhibited by antibody to WapA or AgA (P<0.001). The results obtained confirmed the collagen-binding activity of CBD in AgA and WapA, and suggested that WapA may be used, not only as a vaccine against coronal and dental root caries, but also against S. mutans-mediated endocarditis.