Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood.

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34-99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72-96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57-57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12-100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72-96.27), comparable to test results from untreated samples. The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

Development and evaluation of an M2-293FT cell-based flow cytometric assay for quantification of antibody response to native form of matrix protein 2 of influenza A viruses.

Matrix protein 2 (M2) of influenza A viruses is an attractive target for the development of broadly cross-protective influenza vaccines and therapeutic antibodies. The available evidence suggests that antibodies reactive to the natural tetrameric form of M2 proteins, rather than those to synthetic peptides of M2 ectodomain (M2e), best correlate with M2-mediated immune protection. However, the current ability to quantify strain-specific and/or subtype-cross-reactive M2 antibodies against the natural form of M2 antigens from influenza A viruses of different host origin is limited. In the present study, we generated a panel of 293FT transfected cell lines stably expressing full-length tetrameric forms of M2 molecules from human, avian and the swine-origin 2009 pandemic H1N1 influenza A virus, respectively, and developed an M2-293FT cell line-based flow cytometric assay (M2-FCA). Side-by-side comparison of M2-FCA with a synthetic M2e peptide-based indirect ELISA (M2e-ELISA) reveals that M2-FCA is highly efficient in quantifying both M2e sequence-specific and cross-reactive antibodies to the native form of M2 antigens. In contrast, promiscuity was evident when specificity and cross-reactivity of anti-M2 antibodies were assessed by M2e-ELISA. These results demonstrate that M2-FCA represents a rapid, simple and sensitive method to quantitatively assess specificity and cross-reactivity of anti-M2 antibodies after infection or vaccination.

Comparative analysis of direct fluorescence, Zenon labeling, and quantum dot nanocrystal technology in immunofluorescence staining.

A comparative analysis was performed to determine the sensitivity and efficiency of three fluorescent labeling techniques, including direct fluorescent-antibody staining (FA), Zenon labeling, and quantum dot (QD) nanocrystal technology. Two varicella-zoster virus immunoglobin (Ig) G forms, mAb 4F9 and mAb g62, were selected for these studies. The results indicated that: (1) All three methods demonstrated similar brightness and photostability; (2) the time required to conjugate the antibody varied, with Zenon labeling being the quickest; and (3) the stability of each conjugated complex was different, with FITC/rhodamine-conjugated antibody being the most stable.

Stability of varicella-zoster virus and herpes simplex virus IgG monoclonal antibodies.

The stability of 3 monoclonal antibodies was analyzed at various temperatures and freeze/thaw cycles. Two varicella-zoster virus (VZV) IgGs (mAb 4F9 and mAb g62) and 1 herpes simplex virus 1 (HSV-1 mAb 1D4) were selected for these studies. IgGs were either incubated at various temperatures (25 degrees C, 37 degrees C, 45 degrees C, and 60 degrees C) for different periods of time (0 to 9 weeks) or processed for several freeze/thaw cycles. The reactivities of mAbs 4F9 (IgG1), g62 (IgG1) and 1D4 (IgG2b) were tested by indirect immunofluorescence assay (IFA). The results indicated that: (1) all three mAbs were stable at 25 degrees C and 37 degrees C for 9 weeks; (2) although the reactivities of mAbs g62 and 1D4 were diminished after 5 weeks, mAb 4F9 was stable at 45 degrees C for 9 weeks; and (3) all 3 IgGs lost reactivity after overnight incubation at 60 degrees C. In addition, the results showed that the reactivity of mAbs 4F9, g62 and 1D4 was not diminished after 12 freeze/thaw cycles.

Stability of varicella-zoster virus open reading frame 63.

The stability of varicella-zoster virus (VZV) open reading frame (ORF) 63 was analyzed by sequential passage of a virus strain in cell culture. VZV was propagated in culture for 1,206 passages. ORF63 from six passages (18, 220, 516, 730, 1060, and 1,206) was selected and sequenced. Among the six passages, only passage 1,206 showed point mutations at three locations: 551, 618 and 661. In addition, western blot analysis with anti-ORF63 monoclonal antibodies showed no discernable difference in the size of the ORF63 gene product from passage 18 and that from passage 1,206. These results indicate the stability of VZV ORF63 gene in culture over 1,206 passages.

Rapid identification and authentication of closely related animal cell culture by polymerase chain reaction.

Animal cell lines are important resources for research and diagnostic applications. Cross-contamination and misidentification of cell lines, however, can cause major problems for research (for example, false results that come from contamination cells may mislead the science). Hence, it is imperative to routinely monitor cell lines for identity and authenticity. Here, we extend our previous work on identification and authentication of animal cell culture by polymerase chain reaction (PCR) amplification and DNA sequencing. A PCR-based method for rapid identification and authentication of closely related cell lines was described. In this method, two new primers were designed based on high homology in the aldolase gene family. Used together with our previous primers, the combinations of primers were able to differentiate closely related species, including human from monkey and mouse from rat. This PCR assay provides a rapid, simple, sensitive, and cost-effective method for authentication of closely related cell lines.

Morphologic, immunologic, and molecular methods to detect bacillus anthracis in formalin-fixed tissues.

Due to the importance of Bacillus anthracis as a cause of naturally occurring infection among humans and as an agent of bioterrorism, there is a vital need for rapid and specific assays, including immunohistochemistry (IHC) and polymerase chain reaction (PCR) assays, to detect the bacterium in formalin-fixed tissues. Colorimetric IHC assays were developed using a multistep indirect immunoalkaline phosphatase method with anti-B. anthracis cell wall (EAII-6G6-2-3) and anti-B. anthracis capsule (FDF-1B9) mAbs to detect B. anthracis antigens in formalin-fixed, paraffin-embedded bacterial cultures and tissues. B. anthracis antigens were localized, using both antibodies, in samples from B. anthracis-infected animals and humans. The colorimetric IHC assay with both antibodies was expedient in diagnosing the presence of B. anthracis in formalin-fixed, paraffin-embedded tissue from bioterrorism-associated cases of inhalational and cutaneous anthrax and from a case of naturally occurring cutaneous anthrax. Using the same antibodies, confocal microscopy demonstrated the structure of replicating B. anthracis in tissues. B. anthracis-specific primers were successfully used with PCR to amplify and detect B. anthracis sequences derived from formalin-fixed tissues of anthrax cases. In this study, morphologic, immunologic, and molecular assays were used to study and diagnose 22 veterinary and human anthrax cases.

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing.

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.

Therapeutic applications of monoclonal antibodies.

Researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. However, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. Chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. Antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. Monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases.