Sample
Human serum or plasma
Intended Use
The Human Anti-Rift Valley Fever Virus Nucleoprotein (RVFV NP) IgG ELISA Kit is an immunoassay suitable for quantifying IgG antibody activity specific for RVFV NP in serum or plasma of vaccinated, immunized and/or infected hosts.
This immunoassay is suitable for:
1. Determining immune status relative to non-immune controls.
2. Assessing efficacy of vaccines, including dosage, adjuvantcy, route of immunization and timing.
3. Qualifying and standardizing vaccine batches & protocols.
The assay is for research use only (RUO) and is not intended nor validated for diagnosing Rift Valley Fever virus disease. Reagents contain no virus or viral antigens.
Contents of Kit
| Component | Amount | Contents |
| RVFV NP Coated Strip Plate | 8-well strips (12) | Coated with purified recombinant RVFV NP, and post-coated with stabilizers. |
| Anti-RVFV NP Calibrators: 1 U/mL; 2.5 U/mL; 5 U/mL; 10 U/mL | 0.65 mL each | Four (4) vials, each containing anti-RVFV NP in buffer with antimicrobial as stabilizers. |
| Anti-RVFV NP Positive Control | 0.65 mL | Antiserum with anti-RVFV NP reactivity; value range is on the label. |
| Low NSB Sample Diluent (LNSD) | 30 mL | Buffer with protein, detergents and antimicrobial. Reduces non-specific binding. Use as is for sample dilution. |
| TMB Substrate | 12 mL | Chromogenic substrate for HRP containing TMB and peroxide. |
| Stop Solution | 12 mL | Dilute sulfuric acid. |
| Wash Solution Concentrate (100x) | 10 mL | To be reconstituted: dilute the entire volume (10 mL + 990 mL) with distilled or deionized water into a clean stock bottle; label as Working Wash Solution. |
| Sample Diluent Concentrate (20x) | 10 mL | To be reconstituted: dilute the entire volume (10 mL + 190 mL) with distilled or deionized water into a clean stock bottle; label as Working Sample/Conjugate Diluent. |
| Anti-Human IgG-HRP Conjugate Concentrate (100x) | 0.15 mL | Peroxidase conjugated anti-human IgG in buffer with detergents and antimicrobial as stabilizers. Dilute fresh as needed. |
| Product Manual | 1 | Instruction manual. |
Performance Characteristics
Example - Human Serum/Plasma IgG: A panel of sera from individuals with unknown history was tested for anti-RVFV NP IgG (1:200 dilution in Low NSB Sample Diluent). Threshold Index was calculated using the 1 U/mL Calibrator.
Results: Anti-RVFV NP IgG: ten (10) samples were negative (below the 1.0 threshold); two other samples (E, F) were borderline at 1:200 dilution.
Notes:
1. Positives may be due to prior encounter with the virus or non-RVFV proteins with common epitopes, or may be an aspect of the innate immune repertoire.
2. When the Positive Index is above 5.0, using a dilution curve to calculate titer is a more accurate quantitation method.
3. The sensitivity of the assay may be adjusted by changing the sample dilutions.
RVFV NP Protein: Recombinant RVFV NP protein was expressed in HEK 293 as his-tag fusion protein (a.a. 1-245), accession # P21700.
Antibody Specificity: The anti-human IgG-HRP used in the kit is specific for human IgG with no significant detection of IgM, IgA, IgE or other antibodies. This kit is optimized for human samples.
Detection Range
Calibrators: 1 - 10 U/mL
Sensitivity
The sensitivity of the assay to detect anti-RVFV NP IgG, from either natural exposure or vaccination, is controlled so that the 1 U/mL Calibrator represents a threshold OD for most true positives in human serum diluted to 1:200 or greater.
General Description
Rift Valley Fever virus (RVFV) is a mosquito borne zoonotic disease endemic to Africa. RVFV is a negative stranded RNA virus which belongs to the Bunyaviridae family and Phlebovirus genus. RVFV is characterized by high rates of abortion in ruminants and hemorrhagic fever, encephalitis, and blindness in humans. The RVFV genome encodes 7 proteins: the nucleoprotein (NP), M, NSs, NSm, Gn, Gc, and the L protein.
Sheep, goats, camels, and cattle are the most susceptible animals affected by RVFV. Humans can become infected by RVFV through contact with aerosols of body fluids from infected animals or from the bites of mosquitoes. RVFV is classified as a category A priority pathogen by the NIH and an overlap select agent by the HHS and USDA. Currently a live-attenuated MP-12 vaccine generated from the pathogenic wild-type strain ZH548 is conditionally licensed for use for veterinary purposes in the United States. RVFV is one of several viruses identified by WHO as a likely cause of future epidemics in a new plan developed after the 2014 Ebola epidemic for urgent research and development toward new diagnostic tests, vaccines, and medicines. Current diagnosis of RVFV relies on viral isolation from tissues or serological testing using an ELISA.
Standard Curve
Human Anti-RVFV NP IgG - Calibrators, Controls and Samples (example): dilution curve of an anti-RVFV NP antibody, derived from NP immunization (Calibrators: 1, 2.5, 5 and 10 U/mL), showing the OD range of the assay; the dashed "Cut-off" line indicates the 1 U/mL threshold distinguishing between Positive/Negative.
Reconstitution And Storage
The microtiter well plate and all other reagents, if unopened, are stable at 2 - 8 °C until the expiration date printed on the box label.
Stabilities of the working solutions:
• Working Wash Solution: store at 4 °C for long term and ambient temperature for short term.
• Working Sample/Conjugate Diluent: store at 2 - 8 °C until the kit lot expires or is used up.
• Diluted Anti-Human IgG-HRP Conjugate: use within the working day and discard; return the 100x concentrate to 2 - 8 °C storage.
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