GTCDxᵀᴹ Rat Anti-AAV2 antibody ELISA Kit | Creative Diagnostics DEIASL347R product information

product summary

company name :

Creative Diagnostics

product type :

ELISA/assay

product name :

GTCDxᵀᴹ Rat Anti-AAV2 antibody ELISA Kit

catalog :

DEIASL347R

quantity :

96T

more info or order :

Creative Diagnostics product webpage

product information

Catalog :

DEIASL347R

proudctName :

GTCDxᵀᴹ Rat Anti-AAV2 antibody ELISA Kit

common :

AAV2

Short Name :

GTCDxᵀᴹ Rat Anti-AAV2 antibody ELISA Kit

Detection Method :

ELISA

Common name :

AAV2

Species :

Rat

Application :

Quantitative or qualitative

Sample :

Serum

Size :

96T

Storage :

1. All reagents should be stored at 2°C to 8°C for stability. 2. All the reagents and wash solutions should be used within 12 months from manufacturing date. 3. Before using, bring all components to room temperature (18-25°C). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions. 4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.

Target :

AAV2

Species Reactivity :

Rat

Intended Use :

The Rat Anti-AAV2 ELISA is used as an analytical tool for qualitative determination of antibodies to AAV2 in rat serum.

General Description :

Researchers have used AAV-based vectors in pre-clinical research and in clinical trials in which AAV-based vectors have demonstrated a good safety profile. AAVs have also demonstrated lasting therapeutic gene expression following a single treatment in preclinical and clinical studies. One of the major challenges in AAV-based gene therapy is the presence of circulating anti-AAV antibodies, which can pre-exist in patients and may prevent successful gene transfer. High levels of circulating anti-AAV antibodies can develop after a single administration of gene therapy and can prevent successful gene transfer in patients.

Principle Of The Test :

The qualitative immunoenzymatic determination of specific antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique. AAV2 capsid protein is precoated onto microwells. Samples and control are pipetted into microwells and antibodies to AAV2 present in the sample are bound by the capture protein. After incubation, washing is done to remove the unbound Anti-AAV2. An enzyme linked polyclonal antibody specific for rat IgG is pipetted and incubated. After washing microwells in order to remove any non-specific binding, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of antibodies to AAV2 in the sample. Color development is then stopped by addition of stop solution. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

Reagents And Materials Provided :

1. AAV2 Capsid Coated Microtiter Plate, 12 × 8 wells 2. Negative Control, ready to use,1 × 1 ml 3. Anti-AAV2 Positive Control, ready to use,1 × 1 ml 4. Anti-rat IgG Conjugate, ready to use, 1 × 12 ml 5. Sample Diluent, ready to use, 1 × 50 ml 6. Wash Buffer, 20 × concentrate, 1 × 50 ml 7. TMB Substrate, ready to use, 2 × 6 ml 8. Stop Solution, ready to use, 1 × 7 ml 9. Instruction Manual

Materials Required But Not Supplied :

1. Microtiter Plate Reader able to measure absorbance at 450 nm, with the correction wavelength set at 620 nm 2. Adjustable pipettes and multichannel pipettor to measure volumes ranging from 25 ul to 1000 ul 3. Deionized (DI) water 4. Wash bottle or automated microplate washer 5. Graph paper or software for data analysis 6. Timer 7. Absorbent Paper

Specimen Collection And Preparation :

Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifugation for 10-15 minutes at 1500 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Lipaemic, hemolytic or contaminated samples should not be run. Sample Preparation Before Use Test Sample preparation- allow samples to reach room temperature prior to assay. Take care to agitate samples gently in order to ensure homogeneity. Samples have to be diluted 1 in 10 (v/v), e.g. 30 ul sample in 270 ul sample diluent, prior to assay. The samples may be kept at 2 - 8°C for up to three days. Long-term storage requires -20°C.

Plate Preparation :

A typical Assay Setup * All controls and samples are run in duplicates P.C – Positive control wells N.C – Negative control wells S. – Sample wells

Reagent Preparation :

1. Label any aliquots made with the kit Lot No and Expiration date and store it at appropriate conditions mentioned. 2. Bring all reagents to Room temperature before use. 3. To make Wash Buffer (1X); dilute 50 ml of 20X Wash Buffer in 950 ml of DI water.

Assay Procedure :

Procedural Notes: (1) In order to achieve good assay reproducibility and sensitivity, proper washing of the plates to remove excess un-reacted reagents is essential. (2) High Dose Hook Effect may be observed in samples with very high concentrations of Anti-AAV2. High Dose Hook Effect is due to excess of antibody for very high concentrations of Anti-AAV2 present in the sample. If Hook Effect is possible, the samples to be assayed should be diluted with a compatible diluent. Thus if the Anti-AAV2 concentration of the undiluted sample is less than the diluted sample, this may be indicative of the Hook Effect. (3) Avoid assay of Samples containing Sodium Azide (NaN3), as it could destroy the HRP activity resulting in under-estimation of the amount of Anti-AAV2. (4) It is recommended that all Controls and Samples be assayed in duplicates. (5) Maintain a repetitive timing sequence from well to well for all the steps to ensure that the incubation timings are same for each well. (6) If the Substrate has a distinct blue color prior to use it may have been contaminated and use of such substrate can lead to compromisation of the sensitivity of the assay. (7) The plates should be read within 30 minutes after adding the Stop Solution. (8) Make a work list in order to identify the location of Controls and Samples. Assay Steps: 1. It is strongly recommended that all Controls and Samples be run in duplicates or triplicates. All steps must be performed at 37°C. 2. Add 100 ul of Rat Anti-AAV2 Positive Control, Negative Control, and diluted Samples into respective wells. 3. Cover the plate and incubate for 30 minutes at 37°C. 4. Aspirate and wash plate 5 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step. 5. Pipette without delay in the same order 100 ul of Anti-rat IgG Conjugate into each well. 6. Cover the plate and incubate for 30 minutes at 37°C. 7. Aspirate and wash plate 5 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step. 8. Add 100 ul of TMB Substrate in each well. 9. Incubate the plate for 15 minutes at 37°C in dark. Positive wells should turn bluish in color. (DO NOT SHAKE or else it may result in higher backgrounds and worse precision). 10. Pipette out 50 ul of Stop Solution. Wells should turn from blue to yellow in color. 11. Measure the absorbance at 450/620 nm within 30 min after addition of the Stop Solution.

Calculation :

Determine the Mean Absorbance for each set of duplicate control and Samples. Calculate average (Avg) OD: Avg OD = Average OD from AAV2 Capsid Coated Microtiter Plate Avg OD of the Rat Anti-AAV2 Positive Control should be ≥ 0.5. Avg OD of the Negative Control should be CUT OFF value = Negative Control + 0.1

Interpretation Of Results :

If the Positive Control value is ≥ 0.5, and the Negative Control is If Avg OD of Samples If Avg OD of Samples ≥ CUT OFF, the test samples are considered positive

Precautions :

1. This Kit is For Research Use only. Follow the working instructions carefully. 2. The expiration dates stated on the kit are to be observed. The same relates to the stability stated for reconstituted reagents. 3. Do not use or mix reagents from different lots. 4. Do not use reagents from other manufacturers. 5. Avoid time shift during pipetting of reagents. 6. All reagents should be kept at 2 - 8 °C before use in the original shipping container. . Since the kit contains potentially hazardous materials, the following precautions should be observed - Do not smoke, eat or drink while handling kit material - Always use protective gloves - Never pipette material by mouth - Wipe up spills promptly, washing the affected surface thoroughly with a decontaminant. 8. In any case GLP should be applied with all general and individual regulations to the use of this kit.

applicatons :

The Rat Anti-AAV2 ELISA is used as an analytical tool for qualitative determination of antibodies to AAV2 in rat serum.

Contents of Kit :

Tetramethylbenzidine :