Anti Yellow Fever Virus IgG ELISA

Sample

Serum, plasma

Species Reactivity

Human

Intended Use

Enzyme immunoassay (ELISA) for the determination of IgG antibodies to Yellow Fever Virus (YFV) in human plasma and sera coming from naturally infected people and vaccinated subjects to monitor the presence of protective IgG.

Contents of Kit

The kit contains reagents for the standard configuration of 96 tests.
1. Microplate: MICROPLATE
12 strips × 8 microwells coated with YFV specific native antigens. Plates are sealed into a bag with desiccant.
2. Negative Control CONTROL -
1 × 2.0 mL/vial. Ready to use control.
It contains 1% goat serum proteins, 10 mM Na-citrate buffer pH 6.0 +/-0.1, 0.5% Tween 20, 0.09% Na-azide and 0.1% Proclin-300 as preservatives.
The control is negative for anti YFV antibodies and is yellow colour coded.
3. Positive Control CONTROL +
1 × 2.0 mL/vial. Ready to use control.
It contains 1% goat serum proteins, inactivated human anti YFV IgG, 10 mM Na-citrate buffer pH 6.0 +/-0.1, 0.5% Tween 20, 0.09% Na-azide and 0.1% Proclin 300 as preservatives.
The Positive Control contains anti WNV antibodies and is green colour coded.
4. Wash buffer concentrate: WASHBUF 20×
1 × 60 mL/bottle 20× concentrated solution.
Once diluted, the wash solution contains 10 mM phosphate buffer pH 7.0+/-0.2, 0.05% Tween 20 and 0.05% Proclin-300.
5. Enzyme conjugate: CONJ
1 × 16 mL/vial. Ready to use and red colour coded.
It contains Horseradish peroxidase conjugated polyclonal antibodies to human IgG, 5% BSA, 10 mM Tris buffer pH 6.8+/-0.1, 0.1% Proclin-300 and 0.02% gentamicine sulphate as preservatives.
6. Chromogen/Substrate: SUBS TMB
1 × 16 mL/vial.
It contains 50 mM citrate-phosphate buffer pH 3.5-3.8, 4% dimethylsulphoxide, 0.03% tetramethylbenzidine (or TMB) and 0.02% hydrogen peroxide (or H₂O₂).
Note: To be stored protected from light as sensitive to strong illumination.
7. Sulphuric Acid: H₂SO₄ 0.3 M
1 × 15 mL/vial. It contains 0.3 M H₂SO₄ solution.
Attention: Irritant (H315, H319; P280, P302+P352, P332+P313, P305+P351+P338, P337+P313, P362+P363).
8. Specimen Diluent: DILSPE
2 × 60 mL/vial.
It contains 2% casein, 10 mM Na-citrate buffer pH 6.0 +/-0.1, 0.2% Tween 20, 0.09% Na-azide and 0.1% Proclin-300 as preservatives.
To be used to dilute the sample.
9. Plate sealing foils n°2
10. Instructions for Use n°1

Performance Characteristics

Evaluation of Performances has been conducted on selected panels collected both from (a) vaccinated recipients and healthy individuals living a still YFV almost free region (Italy), from (b) commercial suppliers of certified YFV IgG positive specimens and from (c) a random population of about 600 samples collected in a YFV-endemic country of South America.
1. Specificity and Sensitivity
Specificity is defined as the probability of the assay of scoring negative in the absence of specific analyte.
This parameter was studied on more than 800 samples collected from blood donors and healthy individuals in Italy, country that counts a quite large population of people immigrated from YFV endemic countries, and referring to a commercially available ELISA.
All the samples turned out to be positive in the assay were tested on the reference kit. Those positive results that were not confirmed by the reference kit were considered false positive in the calculation of specificity.
In this study a value > 98% was found.
A panel of YFV IgG negative potentially interfering samples (hemolized, lipemic, etc.) were examined; no interference was observed.
No false reactivity due to the method of specimen preparation has been observed. Both plasma, derived with different standard techniques of preparation (citrate, EDTA and heparin), and sera have been used to determine the value of specificity.
Sensitivity is defined as the probability of the assay of scoring positive in the presence of specific analyte.
The sensitivity was assessed on a panel of samples derived from vaccinated individuals and people living in a YFV-endemic country, totally composed of more than 50 specimens verified positive in the reference ELISA. In this study a sensitivity value > 98% was found.
In summary:
Sensitivity > 98 %
Specificity > 98 %
2. Flavi/Arbo Viruses Cross-Reaction
As expected by the high degree of genetic homology among the viruses bearing to the families Arbo/Flavi, some degree of cross-reaction was observed mostly due to Dengue and Zika Viruses IgG positive samples, collected in Brazil, country quite affected by such viruses. However, as the samples used were not tested for YFV IgG by the supplier such crossreactions cannot be precisely identified and quantified.
3. Precision
It was calculated on three samples examined in replicates in different runs. CV% ranging 2-16% depending on OD450 nm values were seen.

General Description

Yellow Fever virus is an RNA Flavivirus of the family of Flaviviridae transmitted by the mosquito "Aedes Aegypti", causing a severe infection in humans, quite often lethal if not immediately treated. Since many years a very efficient vaccine has been available and widely used to protect people living in endemic regions and travelers.
Diagnosis of YFV natural infection and follow-up of vaccination is generally obtained through detection of virus-specific IgG antibodies by means of ELISA techniques.