ELISA/assay

HHV-6 IgG ELISA Kit

DEIA3565

96T

HHV-6 IgG ELISA Kit

HHV6

HHV-6 IgG ELISA Kit

iELISA

HHV6

Human

Qualitative and semiquantitative

serum or plasma

96T

The ELISA kit should be used within three months after opening. Store the kit and the kit reagents at +2 to +10°C, in a dry place and protected from the light. Store unused strips in the sealable pouch and keep the desiccant inside. Store serum samples at +2 to +10°C up to one week. For longer period make aliquots and keep them at -20°C. Avoid repeated thawing and freezing. Do not store diluted samples. Always prepare fresh. Kits are shipped in cooling bags, the transport time of 72 hours have no influence on expiration. If you find damage at any part of the kit, please inform the manufacturer immediately. Expiration date is indicated at the ELISA kit label and at all reagent labels.

In this test, lysate from infected cells is used as antigen. It contains a set of native viral proteins, some of which are structurally similar to proteins of other herpes viruses, as well as proteins from human cells. Tests of specificity did not show a significant influence of cross-reactivity of IgG antibodies against other herpes viruses on the diagnostic efficiency of ELISA, however, it cannot be completely excluded, especially in samples with a high proportion of low-avid antibodies against group β herpesviruses. Some autoantibodies can also cause false positivity, so test results must be interpreted with caution, especially in patients with autoimmune diseases, transplants, or burns. This test does not differentiate between HHV-6 type A, B and HHV-7 antibodies. In patients with different types of acute infections (CMV, EBV, measles, rubella) a parallel rise of IgG antibodies against HHV-6 was observed. However, the cause was not cross-reactivity, but polyclonal activation of the humoral immune response, or secondary reactivation of HHV-6.

HHV6

Human

Herpes Virus 6; Herpes Virus Type 6; HHV-6

For the detection of IgG antibodies to HHV-6 in human serum isolated from venous or capillary blood.

The kits is intended for serological diagnosis of diseases associated with HHV–6 infection, such as exanthema subitum, acute respiratory illnesses, diarrhoea with fever and febrile seizures in infants, heterophile antibody-negative infectious mononucleosis in children, also interstitial pneumonia, encephalitis, meningitis, hepatitis and aplastic anemia in immunodeficient patients. The presence of IgG anti-HHV-6 antibody reveals the immune status of the patient. Significant rise in anti-HHV-6 IgG antibodies in paired serum samples, taken in acute and convalescent phase of the infection, is indicative of the active infection. The test does not differentiate between HHV-6 subtype A and B.

The anti-HHV-6 IgG is a solid-phase immunoanalytical test. The strips are coated with native HHV-6 antigen. If antibodies are present in the test samples, they will bind to the immobilized proteins. The bound antibodies then react in the next step with horseradish peroxidase-labeled anti-human IgG antibodies. The amount of bound labeled antibodies is determined by a color enzymatic reaction. Negative samples do not react, a slight change in the color of the wells is the background of the reaction.

1. ELISA break-away strips in the handling frame coated with the specific antigen, STRIPS Ag, 1 x 12 pcs 2. 1.3 mL Standard A = Negative control human serum, ready to use. ST A/NC, 1 vial 3. 2.0 mL Standard D = Calibrator (human serum), ready to use. ST D/CAL, 1 vial 4. 1.3 mL Standard E = Positive control human serum, ready to use. ST E/PC, 1 vial 5. 13 mL Anti-human IgG animal antibodies labelled with horseradish peroxidase (anti-IgG Px conjugate), ready to use. CONJ, 1 vial 6. 55 mL Wash buffer, 10× concentrated. WASH 10×, 1 vial 7. 60 mL Dilution buffer, ready to use. DIL, 1 vial 8. 13 mL Chromogenic substrate TMB, ready to use. (TMB/H 2 O 2 ) TMB, 1 vial 9. 13 mL Stop solution, ready to use. (0.4 M sulfuric acid). STOP, 1 vial Notice: Control sera may be colorless to yellowish or blue due to the use of different diluents.

1. Distilled or deionised water for dilution of the Wash buffer concentrate. 2. Appropriate equipment for pipetting, liquid dispensing and washing. 3. Spectrophotometer/colorimeter (microplate reader – wavelenght 450 nm).

a. Allow all kit components to reach room temperature. Turn on the thermostat to 37 °C. b. Thoroughly mix Dilution buffer DIL, Conjugate anti-IgG Px CONJ and Chromogenic substrate TMB. c. Thoroughly mix tested samples and control sera just prior to testing. In the case of a manual test, dilute the test samples 101x with Dilution buffer DIL (eg 5 μL sample + 500 μL Dilution buffer). Do not dilute control sera and calibrator, they are in working concentration (r.t.u., ready to use). d. Prepare a working concentration of Wash buffer WASH 10x by diluting it 10× in a suitable volume of distilled/deionized water (eg. 50 mL of WASH 10× + 450 mL H 2 O). If there are salt crystals in the concentrated solution, warm it in a water bath of + 32 °C to + 37 °C and mix well before diluting. Unused wash solution in working concentration can be stored for 1 month at room temperature. e. Do not dilute Conjugate anti-IgG Px CONJ, Chromogenic substrate TMB and Stop solution STOP, they are ready to use.

The manufacturer is not responsible for the correct function of the kit if the assay procedure is not followed. 1. Allow strips STRIPS Ag, vacuum sealed with desiccant, to reach room temperature before opening the bag, to avoid dew condensation of the plate. Prepare the required number of strips for the reaction. Seal unused strips together with the desiccant in a zipper bag or seal under vacuum. 2. Fill the wells with 100 μL of Standards and diluted serum samples according to the pipetting scheme (Figure 1). Start with filling the first well with 100 μL of Dilution buffer DIL to estimate the reaction background. Then fill the next two wells with Standard D ST D/CAL, serves as a calibrator. Next well with Positive control serum ST E/PC and next one well with Negative control serum ST A/NC. Fill the remaining wells with diluted samples (S1, S2, S3, . ). It is sufficient to apply samples as singlets, however, if you wish to minimize the laboratory error apply ST D/CAL in triplet and the samples and control sera in doublets. We recommend to include positive reference serum sample (your in-house internal control) into each run to follow the sequence, variability and accuracy of calibration. Incubate 30 minutes (+/- 2 min) at 37 °C. 3. Aspirate the contents of the wells into a safety collection bottle containing a suitable disinfectant. Then wash the wells 4 times with 250 μL of wash solution. Avoid overflowing the solution out of the wells. Aspirate the contents of the wells and tap the plate on an adsorbent paper. 4. Mix thoroughly the vial of anti-IgG Px conjugate CONJ and pipette 100 μL of anti-IgG Px conjugate CONJ into the wells. Incubate 30 minutes (+/- 2 min) at 37 °C. 5. Aspirate the fluid from the wells and wash them with 4 x 250 μL of wash solution. Aspirate and tap. 6. Pipette 100 μL of Chromogenic substrate TMB solution into the wells. Incubate for 15 minutes (+/- 30 sec) in the dark at room temperature. Start measuring the incubation time after pipetting the first strip of the plate. Follow this rule to avoid breaking the time interval. Pipette quickly at regular rhythm, or use a suitable dispenser. Cover the strips with foil, an opaque lid, or keep them in a dark place for the duration of the reaction. 7. Stop the reaction by adding 100 μL of Stop solution STOP. Pipette at the same rate as the Chromogenic substrate TMB so that the enzymatic reaction proceeds in all wells at the same time. Check that there are no bubbles in the wells, if so, gently tap the plate frame to remove them. 8. Measure the intensity of the colour reaction on a spectrophotometer/colorimeter at 450 nm within 20 minutes after stopping the reaction. We recommend using a 620-690 nm reference filter. Figure 1: Scheme of application of samples

First, subtract the absorbance of the well with Dilution buffer DIL (DIL = reaction background) from the calibrator, control sera, and tested samples. If the values of Control sera or tested samples are negative after background subtraction, consider them as zero value. 1. Qualitative orientation evaluation 1. Calculate the mean OD value of the Standard D ST D/CAL from the two wells. If you are applying three Standard D ST D/CAL wells and some of these values differ by more than 20% from the mean, do not use it for calculation and calculate the mean of the remaining two values. 2. Determine the cut-off value by multiplying the mean OD value of the Standard ST D/CAL by the correction factor. The value of the correction factor is stated in the Quality Control Certificate for the given kit lot. 3. Samples with an OD value 110 % cut-off are considered positive. 2. Semiquantitative evaluation Determine Positivity Index for each sample: a. First determine the cut-off value as in the previous evaluation method (See paragraph 8.1, point 2). b. Determine the index value for each sample by dividing the OD of the test sample by the cut-off value. c. Read the appropriate degree of reactivity of the sample (See RESULTS EVALUATION). RESULTS EVALUATION * on the basis of the Positivity Index value it is possible to estimate semiquantitatively the amount of antibodies in the sample Note: A rating of +/- means that the sample is in the gray zone. Repeat the test for this result. If the sample is again in the gray zone after retesting, repeat the test with an alternative method or use a sample from a new sample from the same individual 1-2 weeks later.

Anti-HHV-6 IgG antibodies are anamnestic. They persist for the whole life after the primary infection. The kit can detect cross-reactive antibodies to HHV-7. Significant increase of IgG antibodies can be caused by reactivation of the infection, but could not be always proved due to the recurrent character of the reactivations. For final diagnosis, the results of other laboratory tests and the clinical symptoms of the patient should be taken in consideration.

The kit is intended for the qualitative and semiquantitative detection of anti-HHV-6 IgG antibodies in human serum and plasma. Suitable specimens are serum and plasma (heparinised) samples obtained by standard laboratory techniques. Validity of the test The absorbance value of the Dilution buffer DIL (DIL = reaction background) should be The OD values of the standards / control sera and the ratio of the OD values of the standards ST E/PC / ST D/CAL should be within the ranges stated in the Quality Control Certificate of the lot. The Calibrator and Controls are human sera, and as such they may show inhomogeneity, if their value in the test is significantly different from the values stated in the Certificate of analysis, consult the results with the manufacturer.

The interassay variability (between tests) and the intraassay variability (within the test) were determined by testing samples with different OD values. Intraassay The variation coefficient of intraassay is max. 8 %. It is measured for each particular lot at least on 12 parallels of the same microtiter plate. Interassay The variation coefficient of reproducibility is a maximum of 15 %. It is measured for each lot by comparing the wells of the same sample in several consecutive tests.

Measured values of recovery test for every Lot are between 80-120 % of expected value.

Haemolytic and lipemic samples have no influence on the test results up to concentration of 50 mg/mL of haemoglobin, 5 mg/mL of bilirubin and 50 mg/mL of triglycerides. Nevertheless, such samples can only be tested with reservations.

All ingredients of the kit are intended for laboratory use only. Controls contain human sera that has been tested negative for HBsAg, anti-HIV-1,2 and anti-HCV. However they should be regarded as contagious and handled and disposed of according to the appropriate regulations. Autoclave all reusable materials that were in contact with human samples for 0.5 hour at 121°C, burn disposable ignitable materials, decontaminate liquid wastes and nonignitable materials with 3% chloramine. Liquid wastes containing acid (Stop solution) should be neutralized in 4% sodium bicarbonate solution. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin, rinse immediately with plenty of water and seek medical advice. Do not smoke, eat or drink during work. Do not pipette by mouth. Wear disposable gloves while handling reagents or samples and wash your hands thoroughly afterwards. Avoid spilling or producing aerosol.

a. All kit components are for laboratory use only. b. The manufacturer guarantees the usability of the kit as a whole. c. Work aseptically to avoid microbial contamination of samples and reagents. e. When collecting, diluting, and storing reagents, be careful not to cross-contaminate them or contaminate them with enzymatic activity inhibitors. f. The Chromogenic Substrate TMB shouldn't come into contact with oxidizing agents and metal surfaces. Because it is sensitive to light, close the bottle immediately after use. The Chromogenic substrate TMB must be clear in use. Do not use the solution if it is blue. g. Follow the Instruction manual exactly. Non-reproducible results may arise in particular: insufficient mixing of reagents and samples before use inaccurate pipetting and non-compliance with the incubation times poor washing technique and splashing of the edges of the wells with sample or conjugate using the same tip when pipetting different solutions or swapping caps h. Human control sera and standards used in the kit were tested for the absence of HBsAg, HCV and antiHIV-1,2 antibodies. Treat test specimens, control sera, standards, and used strips as infectious material. Autoclave items that have been in contact with them for 1 hour at 121 °C or disinfect for at least 30 minutes with 3% chloramine solution. i. Neutralize liquid waste containing Stop solution (sulfuric acid solution) with 4% sodium bicarbonate solution before disposal. j. Disinfect the waste generated during strip washing in a waste container using a suitable disinfectant solution (eg Incidur, Incidin, chloramine, . ) at the concentration recommended by the manufacturer. k. Handle Stop solution STOP carefully to avoid splashing on the skin or mucous membranes. If this happens, wash the affected area with plenty of running water. l. Do not eat, drink or smoke while working. Do not pipette by mouth, but by suitable pipetting devices. Wear protective gloves and wash your hands thoroughly after work. Be careful not to spill specimens or form an aerosol. m. All reagents and packaging material must be disposed of in accordance with applicable legislation. n. In case of suspicion of an adverse event in connection with the use of the kit, inform the manufacturer and the competent state authority without delay.

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet