MicroELISA

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The EIAgen Detect HIV Combo Kit is a 4th generation solid phase Enzyme-Linked Immunosorbent assay using a mixture of antigens and antibodies for the in vitro diagnostic screening in human serum or plasma (EDTA, Heparin and Citrate) of antibodies to HIV-1, HIV-2 and HIV-1 p24 antigen. This kit is a combined Ag/Ab assay and is not to be used for the detection of HIV-1 p24 antigen alone. This kit is for in vitro diagnostic use by a health-care professional and will not be sold to the general public.

Acquired immune deficiency syndrome (AIDS) is a set of symptoms resulting from the incapacitation of the human immune system caused by the Human Immunodeficiency Virus (HIV). HIV infection may progress to a symptomatic phase that is characterized by opportunistic infections and that may cause death.

The etiological agent of AIDS, HIV, targets specific types of T cells causing Lymphopenia and affecting T cell mediated immunity. HIV is a member of a retrovirus family with two sub-families: HIV-1 and HIV-2. HIV-1 is more virulent and transmittable than HIV-2.
HIV-1 is the cause of HIV infections globally, whereas HIV-2 is found predominantly in the countries of West Africa. As serological cross-reaction between HIV-1 and HIV-2 is highly variable and dependent on the tested sample, antigens for the specific detection of both HIV-1 and HIV-2 are included in the assay.

HIV is transmitted through sexual contact with infected persons, sharing needles and syringes with infected people and transfusion of contaminated blood.

Enzyme-Linked Immunosorbent Assays (such as the EIAgen Detect HIV Combo Kit ELISA 4th gen) are recommended for screening human blood and plasma for the presence of anti-HIV antibodies and HIV-1 p24 antigen. The presence of anti-HIV-1 and/or anti-HIV-2 in the blood indicates potential infection with HIV-1 and/or HIV-2 and consequently this blood should not be used for transfusion or for manufacture of injectable products.

Antigens representing epitopes of HIV-1 gp41 and HIV-2 gp36 are coated onto microplate wells together with monoclonal antibodies against HIV-1 p24. Serum or plasma sample is added to the well and if antibodies specific for HIV-1 and/or HIV-2 (IgG, IgM or IgA) are present in the sample, stable complexes will be formed with the HIV antigens attached to the well. HIV-1 p24 antigen, if present will bind simultaneously to the antibodies in the well and to the detector antibodies present in the Assay Diluent. Non-reactive antibodies are removed by washing. Stable antigen-antibody complexes are identified through the successive addition of biotinylated antigens and horseradish peroxidase (HRP) conjugated streptavidin. These antibody-antigen complexes are quantified through the catalytic activity of horseradish peroxidase. Peroxidase substrate solution is added and is converted to a blue-coloured product. A positive sample generates a dark blue colour while faint blue colour or colourless wells indicate a negative sample. Upon adding stopping solution, the colour of the solution will change from blue to yellow. Optical Density (OD) is measured with a spectrophotometer (ELISA reader) at 450 nm with reference wavelength at 600-650nm and is in proportion to the amount of anti-HIV1/2-antibodies and HIV-1 p24 present in the sample

The kit contains reagents for 96 tests (code 081311C), or 192 tests (code 081312C) or 480 tests (code 081315C).

1. Microplate

12 strips of 8 breakable wells coated with HIV specific gp36 and gp41 peptides and gp41 protein with Monoclonal Antibodies specific to the HIV-1 p24 Ag.

Plates are sealed into an aluminium pouch with desiccant. Bring the microplate to room temperature (18 - 25°C) before opening the bag. Unused strips have to be returned into the pouch and the pouch has to be sealed and stored back to 2 - 8°C, in presence of the desiccant.

2. Negative Control

Ready to use control. It contains human serum negative for HIV antibodies and for p24 antigen, and 0.05% Proclin 300 as preservative.

3. Positive Control HIV-1

Ready to use control. It contains human serum positive to Anti-HIV 1 and 0.05% Proclin 300 as preservative.

Note: The absence of viable pathogens in the positive control can not be fully ensured, and therefore, the control should be handled as potentially biohazardous, in accordance with good laboratory practices

4. Positive Control HIV-2

Ready to use control. It contains human serum positive to Anti-HIV 2 and 0.05% Proclin 300 as preservative.

Note: The absence of viable pathogens in the positive control can not be fully ensured, and therefore, the control should be handled as potentially biohazardous, in accordance with good laboratory practices.

5. Positive Control HIV p-24

Ready to use control. It contains recombinant HIV-1 p24 and 0.05% Proclin 300 as preservative.

Note: The absence of viable pathogens in the positive control can not be fully ensured, and therefore, the control should be handled as potentially biohazardous, in accordance with good laboratory practices

6. Conjugate # 1

Ready to use reagent. It contains a mixture  biotinylated antigens in phosphate buffer and 0.05% Proclin 300 as preservative.

Code colored with blue dye at 0.004 mg/mL.

Note: When the Conjugate # 1 is added, each well, except the 1^st blanking well, is blue coloured.

7. Conjugate # 2 Concentrate 20X

20x solution concentrate. It contains Horseradish Peroxidase conjugated with streptavidin in specific stabilizer buffer with preservative.

Reagent to be diluted with the Conjugate # 2 Diluent.

Note: Any unused portion of this diluted Conjugate #2 Solution may be stored at 2 - 8°Cfor no more than 6  days.

8. Conjugate # 2 Diluent

Ready to use reagent.

Reagent is used for the dilution of the Conjugate #2 Concentrate 20X. It contains phosphate buffer and 0.05% Proclin 300 as preservative.

Code colored  with orange dye at 0,05 mg/mL.

9. Assay Diluent

Ready to use reagent.

It contains biotinylated Anti HIV-1p24 Ag, mouse serum proteins, human IgG and 0.05% Proclin 300 as preservative.

Code colored with red dye at 50 mM.

Note: The diluent changes colour from yellowish red to brownish red colour in the presence of sample.

10. Substrate TMB

Ready-to-use component. It contains 0.26 mg/mL of 3,3’,5,5’ Tetramethylbenzidin (TMB) and 0.01% w/v of Hydrogen peroxide (H₂O₂), in citrate buffer. 

Mix gently before use.

Note: To be stored protected from light as sensitive to strong illumination.

11. Stop Solution

Ready-to-use component.

It contains 0,3 M H₂SO₄ solution.

Mix gently before use.

12. Wash Buffer Concentrate 25X

25X concentrated solution.

It contains 0.2% Proclin 300 as preservative. Once diluted, the wash solution (wash buffer diluted) contains phosphate buffer saline  and  Proclin 300 and Tween 20 as preservatives.

Once diluted, the wash solution is stable for 6 days at 2 - 8°C.

E. MATERIALS REQUIRED BUT NOT PROVIDED
1. Calibrated Micropipettes of 200 μL, 50 μL and 10 μL and disposable plastic tips. 2. EIA grade water (double distilled or deionised, charcoal treated to remove oxidizing chemicals used as disinfectants). 3. Timer with 60 minute range or higher. 4. Absorbent paper tissues. 5. Calibrated ELISA microplate thermostatic incubator (dry or wet) set at +37°C (± 1°C tolerance). 6. Calibrated ELISA microwell reader with 450 nm (reading) with 620 nm (back ground substraction) and 405 nm filters. 7. Calibrated ELISA microplate washer. 8. Vortex or similar mixing tools.

P. PERFORMANCE CHARACTERISTICS
Evaluation of Performances has been conducted in accordance to what reported in the Common Technical Specifications (CTS) as required by art. 5, Chapter 3 of IVD Directive 98/79/EC.
The performances evaluation was carried out both in an external centre and in Adaltis’ laboratories as well to complete the study.

For more read IFU.

IVD product not available for all countries. Please contact our Sales department (email: sales@adaltis.net) for specific countries enquiries

CE2797