Human Anti-RVG IgG ELISA Kit | Creative Diagnostics DEIAHRVPY28 product information

product summary

company name :

Creative Diagnostics

product type :

ELISA/assay

product name :

Human Anti-RVG IgG ELISA Kit

catalog :

DEIAHRVPY28

quantity :

96T

more info or order :

Creative Diagnostics product webpage

product information

Catalog :

DEIAHRVPY28

proudctName :

Human Anti-RVG IgG ELISA Kit

common :

RABV

Short Name :

Human Anti-RVG IgG ELISA Kit

Detection Method :

iELISA

Common name :

RABV

Species :

Human

Application :

Quantitative

Sample :

Serum, plasma

Size :

96T

Storage :

The unopened kit is stable at 2-8°C until the expiration date printed on the box label. The redissolved standards must be stored at ≤ -20°C and be stable for 3 months.

Species Reactivity :

Human

Intended Use :

The Human anti- Rabies Virus Glycoprotein (RVG) IgG ELISA Kit is an immunoassay suitable for detecting and quantifying IgG antibody activity specific for RVG, in serum or plasma. Other biological fluids, including tissue culture medium, may be validated for use. For research use only (RUO), not for diagnosis, cure or prevention of the disease.

General Description :

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence.

Principle Of The Test :

The Human Anti-RVG IgG ELISA kit is based on the binding of anti-RVG IgG in samples to RVG antigen immobilized on the microwells, and bound antibody is detected by anti-IgG-specific HRP conjugate. After a washing step, chromogenic substrate (TMB) is added and color (blue) is developed, which is directly proportional to the amount of antibody present in the sample. Stopping Solution is added to terminate the reaction (converts blue to yellow), and A450nm is then measured using an ELISA reader. The presence of antibody in samples is determined relative to Calibrators.

Reagents And Materials Provided :

1. RVG Antigen Coated Strip Plate 8-well strips (12) 2. Human Anti-RVG IgG Positive Control 1vail 3. Anti-RVG Calibrator 1 U/ml 1vail 4. Anti-RVG Calibrator 2.5 U/ml 1vail 5. Anti-RVG Calibrator 5 U/ml 1vail 6. Anti-RVG Calibrator 10 U/ml 1vail 7. Anti-Human IgG HRP Conjugate (1000X) 12 μl 8. HRP Diluent 12mL 9. Sample Diluent (2X) 15 ml 10. Wash Buffer (20X) 50 ml 11. Stop Solution 12 ml 12. Product Manual 1 ea

Materials Required But Not Supplied :

1. Pipettors and pipettes that deliver 100μl and 1-10ml. 2. Disposable glass or plastic 5-15ml tubes for diluting samples and Anti-Human IgG HRP Concentrate. 3. Stock bottle to store diluted Wash Solution; 0.2 to 1L. 4. Distilled or deionized water to dilute reagent concentrates. 5. Microwell plate reader at 450 nm wavelength and ELISA plate washer.

Specimen Collection And Preparation :

Serum and other biological fluids may be used as samples with proper dilution to avoid solution matrix interference. For serum, collect blood by venipuncture, allow clotting, and separate the serum by centrifugation at room temperature. For other samples, clarify the sample by centrifugation and/or filtration prior to dilution in Sample Diluent. If samples will not be assayed immediately, store refrigerated for up to a few weeks, or frozen for long-term storage. Caution: Human serum and other bodily fluids may contain infectious material. Always wear gloves when handling human samples, including the standards and controls (which have been tested non-reactive for HBsAg and Anti-HIV), and dispose of these samples and containers as biohazard waste.

Plate Preparation :

Review Interpretation of Results and Limits of the Assay before proceeding: 1. Select the proper sample dilutions accounting for expected potency of positives and minimizing non-specific binding (NSB) and other matrix effects; for example, net signal for non-immune samples should be lower than the 1 U/ml Calibrator. This is usually 1/100 or greater dilution for human serum/plasma with normal levels of IgG and IgM. 2. Run a Sample Diluent Blank. This signal is an indicator of proper assay performance, especially of washing efficacy, and is used for net OD calculations, if required. Blank OD should be 3. Run the Human Anti-RVG IgG Positive Control. 4. Run a set of Calibrators, which validate that the assay was performed to specifications: 10 U/ml should give a high signal (>1.5 OD); 1 U/ml should give a low signal which can be used to discriminate at the Positive/Negative threshold.

Reagent Preparation :

Bring all reagents to room temperature (20-30°C) equilibration (at least 30 minutes). 1. Determine the number of wells for the assay run. Duplicates are recommended, including 8 Calibrator wells and 2 wells for each sample control to be assayed. 2. Remove the appropriate number of microwell strips from the pouch and return unused strips to the pouch. Reseal the pouch and store refrigerated. 3. Use 650μL purified water to re-dissolved the standards and positive control. 4. Sample Diluent(1X): Diluent to working concentration using 15mL purified water. 5. Wash Buffer(1X): If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 25 ml of Wash Buffer Concentrate (20x) into purified water to prepare 1000 ml of Wash Buffer (1x). 6. Anti-Human IgG HRP Conjugate (1x): Centrifuge the vial before opening. Anti-Human IgG HRP Conjugate requires a 1000-fold dilution. According to demand, diluent the Anti-Human IgG HRP Conjugate using the HRP Diluent.

Assay Procedure :

ALL STEPS ARE PERFORMED AT ROOM TEMPERATURE. After each reagent addition, gently tap the plate to mix the well contents prior to beginning incubation. 1. Incubation [100μl – 60 min; 5 washes] Add 100μl of calibrators, samples and controls each to predetermined wells. Tap the plate gently to mix reagents and incubate for 60 minutes. Wash wells 5 times and pat dry on fresh paper towels. As an alternative, an automatic plate washer may be used. Improper washes may lead to falsely elevated signals and poor reproducibility. 2.Incubation [100μl – 30 min; 5 washes] Add 100μl of diluted Anti-Human IgG HRP to each well. Incubate for 30 minutes. Wash wells 5 times as in step 2. 3. Substrate Incubation [100μl – 15 min] Add 100μl TMB Substrate to each well. The liquid in the wells will begin to turn blue. Incubate for 15 minutes in the dark, e.g., place in a drawer or closet. Note: If your microplate reader does not register optical density (OD) above 2.0, incubate for less time, or read OD at 405-410 nm (results are valid). 4. Stop Step [Stop: 100ul] Add 100μl of Stop Solution to each well. Tap gently to mix. The enzyme reaction will stop; liquid in the wells will turn yellow. 5. Absorbance Reading Use any commercially available microplate reader capable of reading at 450nm wavelength. Use a program suitable for obtaining OD readings, and data calculations if available. Read absorbance of the entire plate at 450 nm using a single wavelength within 30 minutes after Stop Solution addition. If available, program to subtract OD at 630nm to normalize well background.

Quality Control :

It is recommended that for each laboratory assay appropriate quality control samples in each run to be used to ensure that all reagents and procedures are correct.

Interpretation Of Results :

A. Antibody Activity Threshold Index Compare Samples to 1 U/ml Calibrator or Internal Control = Positive/Negative Cut-off. Results The sensitivity of the assay to detect anti-RVG IgG, from either natural infection or vaccination, is controlled so that the 1 U/ml Calibrator represents a threshold OD for most true positives in human serum diluted to 1:100 or greater. Visual inspection of the data in the above graph shows the following: Calibrators - dilution curve of an anti-RVG antibody, derived from RVG vaccination, shows the OD range of the assay; high value indicates optimal sensitivity of the assay. 1 U/ml: a "Cut-off" line has been drawn to indicate a threshold distinguishing between Positive/Negative. The is not a clear-cut threshold, rather a low OD area that could represent either low positives or high background negatives. Positive Control -clearly positive (>0.5 net OD) Internal Control -a true positive from an infected patient that represents the lab's experience in distinguishing low positive from negative samples. This should be run in each assay to supplement the 1U/ml Calibrator for Positive/Negative discrimination purposes. The 1 U/ml Calibrator can be used to calculate a Threshold Index that numerically discriminates Positive/Negative: Divide each Sample net OD by the 1 U/ml Calibrator net OD. Values above 1.0 area measure of Positive Antibody Activity; below 1.0 are Negative for antibody. B. Positive Index Experimental sample values may be expressed relative to the values of Control or Non-immune samples, by calculation of a Positive Index. One typical method is as follows: 1. Calculate the net OD mean + 2 SD of the Control/Nonimmune samples = Positive Index. 2. Divide each sample net OD by the Positive Index. Values above 1.0 area measure of Positive Antibody Activity; below 1.0 are Negative for antibody. A sample value would be Positive if significantly above the value of the pre-immune serum sample or a suitably determined non- immune panel or pool of samples, tested at the same sample dilution. This calculation also quantifies the positive Antibody Activity level, assigning a higher value to samples with higher Antibody Activity, and vice versa.

Typical Standard Curve :

U/mL OD1 OD2 10 3.2071 2.9014 5 1.8983 1.6531 2.5 0.8349 0.8083 1 0.4261 0.3861 0 0.0141 0.016 Positive Control 0.6609 0.6484

Precision :

U/mL Intra CV Inter CV 10 7% 8% 5 4% 10% 2.5 3% 8% 1 8% 9%

Sensitivity :

The RVG antigen coating level and HRP conjugate concentration are optimized to differentiate anti-RVG IgG from background (non-antibody) signal with human serum or plasma samples diluted 1:100. Notes The sensitivity of the assay may be increased to perhaps convert a borderline sample to a positive by using a lower dilution of the sample, e.g., 1/50. The values of negatives may increase, so an alternative threshold should be established using known negatives to develop a Positive Index, or by using known Internal Controls as discriminator for a Threshold Control (instead of the kit 1 U/ml Calibrator Control)

Precautions :

Calibrators, Sample Diluent, and Antibody HRP contain bromo nitro dioxane (BND: 0.05%, w/v). Stop Solution contains dilute sulfuric acid. Follow good laboratory practices, and avoid ingestion or contact of any reagent with skin, eyes or mucous membranes. All reagents may be disposed of down a drain with copious amounts of water.

Limitations :

The assay detects and quantifies IgG antibodies directed to the RVG protein. Animals vaccinated or infected with the rabies virus may not produce antibodies specific to RVG. Anti-RVG antibody levels of an infected or vaccinated animal maybe below detection threshold related to the time course of the occurrence, e.g., too early for positive titer development.

applicatons :

Contents of Kit :

Standard Curve :

U/mL OD1 OD2 10 3.2071 2.9014 5 1.8983 1.6531 2.5 0.8349 0.8083 1 0.4261 0.3861 0 0.0141 0.016 Positive Control 0.6609 0.6484

Tetramethylbenzidine :