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- All Subjects: Antibodies
- Creators: Department of Chemistry and Biochemistry
- Creators: Nichols, Cole
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
Emil von Behring researched treatments for the common childhood disease diphtheria in Germany in the 1890s and early 1900s. Diphtheria is a lethal disease that infected approximately 40,000 people in Germany between 1886 and 1888 with a general mortality rate of twenty-five percent. Behring investigated treatment of diphtheria using serum therapy, which is an alternative to vaccination that uses protective agents from other people’s blood to defend a patient against disease. Behring termed those protective agents antitoxins. He received the first Nobel Prize in Physiology or Medicine for his work on serum therapy, which was one of the first Nobel Prizes given in the field of immunology. Additionally, Behring researched active vaccination as another way to protect patients from diphtheria. Behring’s studies lowered the mortality rate of diphtheria in Germany through serum therapy and vaccination, especially since vaccination confers protection to both mother and infant during pregnancy and after birth.
In 2014, Flor M. Munoz and colleagues published “Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial,” hereafter “Tdap Immunization During Pregnancy,” in the Journal of the American Medical Association. The authors conducted a study to determine how Tdap immunization affected the mother and infant’s immune response to the common childhood diseases tetanus, diphtheria, and pertussis. They found that Tdap immunization did not lead to an increased risk of adverse health events. Furthermore, maternal Tdap immunization provided the infant with protective levels of pertussis antibodies after delivery and did not affect the infant differently from the DTaP vaccination series, which is the version of Tdap for young children. The authors’ findings in “Tdap Immunization During Pregnancy” supported the United States Centers for Disease Control and Prevention’s, or CDC’s, recommendation for pregnant women to receive the Tdap vaccine to prevent disease in mother and infant.
In 2014, Flor M. Munoz and colleagues published “Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial,” hereafter “Tdap Immunization During Pregnancy,” in the Journal of the American Medical Association. The authors conducted a study to determine how Tdap immunization affected the mother and infant’s immune response to the common childhood diseases tetanus, diphtheria, and pertussis. They found that Tdap immunization did not lead to an increased risk of adverse health events. Furthermore, maternal Tdap immunization provided the infant with protective levels of pertussis antibodies after delivery and did not affect the infant differently from the DTaP vaccination series, which is the version of Tdap for young children. The authors’ findings in “Tdap Immunization During Pregnancy” supported the United States Centers for Disease Control and Prevention’s, or CDC’s, recommendation for pregnant women to receive the Tdap vaccine to prevent disease in mother and infant.