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RTA Colorimetric Kit: ELISA-Spot or Microarray
Item#: RTA1001
Our Price:
4 Chips For $150
25 Chips For $540


 

View powerpoint presentation on this product:  51_~JIM-allergy.pdf

Main Features »

Zeta-Grip
Colorimetric Protein Array

Storage Instructions
Chips and plates should be stored dry at room temperature or with refrigeration (2–30 ºC) in their original package in a dust-free environment. Reagents should be stored with refrigeration (4–10 °C) but are stable at room temperature for up to 12 hours.

Reagent Materials provided

Chip-open format and ELISA-Spot macro-kits:
· z-Grip™ chips
· Petri dishes
· Blocking solution
· Wash buffer
· NBT/BCIP
· Detection buffer solution
· Positive control

Important Note
This assay is not compatible with any detergent, including Tween 20 and SDS. If you must use detergents in your assay and they cannot be removed by dialysis or some other method, you will not be able to use this assay.


INTRODUCTION

This kit is intended for protein/antigen assays. Proteins are applied to the zeta-Grip surface manually or by a robotic spotting device. The different formats allow for assay development, including transition from handspot to microspot assays. For example, if a robotic printer is not available, 1–5 μL of antigen material can be spotted into wells to determine feasibility. The second stage of assay development may involve the robotic spotting of small volumes of multiple antigens in microtiters wells. This scheme allows for assay development without requiring a robotic spotter. In most cases, assays developed by handspotting are easily scaled to microspotting. In other cases, macrospotting is acceptable.

An advantage of the zeta-Grip system is that the 3-dimensional protein-binding surface has an extremely high and stable antigen-binding capacity. It is recommended that the first step in assay development be a titration of antigen concentrations. Initial starting concentrations should be 10–1,000 higher than that used for a comparable ELISA assay. If the cost of antigenic material is a concern, it is recommended that after the initial trials, one moves to a microspot format (0.5–20 nL) at the same concentration.

Another advantage of the Zeta-Grip colorimetric system is very low background. For most assays and readers, the colorimetric background signal is below the limit of detection. If you observe a background signal, it is suggest that you further optimize the assay conditions (described below).

An additional advantage of this system is realized when the ratio of fluid used for incubations per spot size is maximized.

The combination of high sensitivity and low background can result in an extremely sensitive assay. It is suggested that initial studies involve serial 1:10 dilutions to evaluate the sensitivity of the specific assay in development. If possible, the initial studies should be conducted in parallel with ELISA.


Initial studies can be performed by “hand spotting” 1–3 μL of material. After an assay is developed, printing can be performed using contact or non-contact printing. In some cases, improved spot morphology and protein stability result from printing at 4–10 °C and with 60–80% humidity.


The Zeta-Grip RTA colorimetric array system has been developed for printing antigens and detecting with an antibody (e.g., human serum) as shown in Figure 1. For sandwich arrays ,we recommend the Zeta-Grip RTA avidin surface (in development).



PROCEDURE

Sample preparation and spotting
1. Dilute antigens in printing buffer. Suggestion: use much higher concentrations than used for plating polystyrene; antigen concentrations can be as high as 0.50 mg/mL. Increased antigen concentrations increase assay sensitivity. See printing section below for more details.
2. After printing, allow Zeta-Grip chips to dry (drying at 4–8 °C with 60–80% humidity can improve spot morphology; do not move chips until after spots are dry).
3. Printed slides are stable for 6 months–4 years (depending on the antigen). For storage and processing of slides, place printed Zeta-Grip slides individually into empty incubation dishes (Petri dishes).

Blocking
4. Add 10 mL Blocking Solution to each incubation dish and gently agitate at room temperature or 37 °C for 20–60 min (depending on assay).
5. Add a user-determined amount or titrations in individual wells of sample or control (primary antibody) solution and incubate with gentle agitation for 20–60 minutes. at room temperature or 37 °C.
6. Appropriately discard the sample or control solution.
7. Wash by performing three rinses with Wash Buffer. In some cases, this wash step can be omitted.
8. Empty incubation dishes and appropriately dispose of wash solutions.
9. Add 10 mL Wash Buffer. Add AP-conjugated secondary antibody (suggested dilution range is 1:1,000–5,000) to each incubation dish. Try highest concentration (1:1,000) first.
10. Incubate at room temperature with gentle agitation for 20 or 60 min.
11. Discard the solutions from the incubation dishes.
12. Wash by performing three (up to 10) rinses with Wash Buffer.

Detection
13. Freshly prepare developer by adding 10 μL NBT-BCIP to 1 mL cold Detection Buffer.
14. Add 10 mL developer to each chip. Incubate chips at room temperature for 12 or more minutes. (Note: For quantitative work, pre-determine development time by conducting pilot study. It is important that all chips to be compared are developed for the same amount of time.) Developing too long (> 16 minutes) can result in increased background and signal saturation.
15. Discard the developer and rinse with COLD tap water (holding under the faucet is okay). Optional: Cover the chips with cold water for 2 minutes to stop further color development. Discard water.
16. Air-dry by allowing excess water to run off. Signal will improve when dry. Optional: Use forced air for rapid drying.
17. After drying, scan the slides and analyze data according.


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