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This Package Insert is provided for product evaluation purposes only and is
not intended to be used in place of the Package Insert shipped with the product.

IMMUNO-TEK
ELISA Construction
System

FOR RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC OR CLINICAL USE.

ZMC Catalog # : 0801188
   
INTENDED
USE
  The IMMUNO-TEK ELISA Construction System is supplied for research purposes or for further manufacturing use only.

The IMMUNO-TEK ELISA Construction System is an easy to use kit that enables scientists to rapidly prepare high quality ELISA assays. ELISA assays prepared with the IMMUNO-TEK ELISA Construction System are of comparable quality to those manufactured by the worlds leading immunodiagnostic companies. The kit contains specially screened microtiter plates and all reagents necessary to coat the plates with antigen or antibody. Microtiter plates prepared with the IMMUNO-TEK ELISA Construction System have a low background, high sensitivity and may be stored desiccated for one to two years with no loss of activity.

For convenience, all components of the IMMUNO-TEK ELISA Construction System may be ordered individually from ZeptoMetrix. This is to meet the needs of scientists who wish to further optimize their ELISA assays through the use of special microtiter plates or coating conditions.

   
PRINCIPLE
OF THE
SYSTEM
  Preparation of ELISA microtiter plates involves three major steps: 1) binding antigen or antibody to the plate; 2) blocking non-specific binding sites on the plate; and 3) coating the plate with a stabilizer to allow dry storage of the plates for long periods of time. The IMMUNO-TEK ELISA Construction System contains Zepto-Bind used in the binding step, Zepto-Block used to block non-specific sites on the plate and Zepto-Coat which stabilizes the antigen or antibody applied to the plate. The IMMUNO-TEK ELISA Construction System also provides microtiter plates from lots of plates that have been specially selected for high binding and low background characteristics.
   
REAGENTS
 
 

Materials Supplied

  • ZeptoBind, 60 ml

  • ZeptoBlock, 100 ml
  • ZeptoCoat, 60 ml
  • Microtiter Plates, 5 each
  • Sealable Bags, 5 each

  • Desiccant Pillows, 5 each
  • Plate Sealers, 5 each

Materials Required but Not Supplied

  • Disposable gloves.
  • Validated adjustable micropipets, single and multichannel.
  • Test tubes and racks for preparing specimen and control dilutions.
  • Graduated cylinders and assorted beakers.
  • Validated automatic microplate washer or manual vacuum aspiration equipment.
  • Validated incubator for 37C 1C.
  • Validated microplate reader.
  • Timer.
  • 1% sodium hypochlorite as disinfectant. May be prepared from household bleach.
  • Distilled or deionized water.

 

   
PLATE COATING
PROCEDURES
 

Allow all reagents to reach room temperature before use. 

Step 1: Dissolve antigen or antibody in ZeptoBind. Prepare enough to add 100 l to each well to be coated.

Step 2: Pipet 100 l of antigen or antibody into each microtiter well.

Step 3: Cover plate with a plate sealer and incubate plate overnight at 4 C.

Step 4: Aspirate antigen or antibody.

Step 5: Pipet 200 l of ZeptoBlock into each microtiter well.

Step 6: Cover microplate with a plate sealer and incubate for 2 hours at room temperature.

Step 7: Aspirate ZeptoBlock.

Step 8: Pipet 100 l of ZeptoCoat into each microtiter well.

Step 9: Cover with a plate sealer and incubate 1 hour at room temperature.

Step 10: Aspirate ZeptoCoat and allow plate to dry for 4 hours at room temperature.

Step 11: Place plate and one desiccant pillow into a resealable bag and store plate until use. Plates prepared by this method will be stable for 12 to 24 months.

   
VARIABLES IN
MICROTITER
PLATE
PRODUCTION
  Although relatively good, general purpose immunoassays can be prepared rapidly by using the reagent manufacturers suggested working concentrations, creation of a high quality immunoassay involves a series of experiments to determine the exact amount of each reagent to be used. Also, in many cases a manufacturers working concentration is not available as is the situation where a scientist has prepared his/her own antigen or antibody. A discussion of many of such optimization procedures can be found in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. This manual also describes a number of different immunoassay formats and architectures. The discussion below is limited to optimizing plate coating conditions using the IMMUNO-TEK Construction System.
   
OPTIMIZATION
OF ANTIGEN
OR ANTIBODY
CONCENTRATION
  The concentration of antibody or antigen will affect the quality of the assay being developed. For most antibodies or antigens, the optimal coating concentration will be in the range of 1 to 10 g/ml when used with ZeptoBind. If the optimal concentration is not known, and it is not practical to perform a titration to determine the amount to use, we recommend using 10 g/ml. This will work well for most applications.

When possible, we recommend titrating the amount of antigen or antibody to be used in plate coating. We also recommend titrating the amout of antigen or antibody when using new lots of materials as there may be significant lot-to-lot variations in antigen or antibody preparations.

In such titrations, increasing amounts of antigens or antibodies will increase the signal, eventually reaching a plateau. Ideally, for an optimal assay one wishes to be at this plateau. Concentrations of antigen or antibody above that needed to achieve a plateau are not only wasteful but in some assays can lead to unacceptable backgrounds in the assay. For example, antigen concentrations from 1 to 10 g/ml may yield steadily increasing signal but concentrations above 10 g/ml show no further signal increase. In this case, 10 g/ml would represent the plateau and the optimal coating concentration would be 10 g/ml.

   
PROCEDURAL
FLOW CHART
 

 DISSOLVE ANTIGEN OR ANTIBODY IN ZEPTOBIND

ADD 100 l TO EACH WELL

INCUBATE OVERNIGHT AT 4   C

ASPIRATE

ADD 200 l ZEPTOBLOCK TO EACH WELL

INCUBATE 2 HOURS AT ROOM TEMPERATURE

ASPIRATE

ADD 100 l ZEPTOCOAT TO EACH WELL

INCUBATE 1 HOUR AT ROOM TEMPERATURE

ASPIRATE

DRY PLATE OVERNIGHT AT ROOM TEMPERATURE

PLACE PLATE IN BAGGIE WITH ONE DESICCANT PILLOW

STORE AT 4 C

   

Rev 09/00

 

For more information :: Bioxys and Gentaur BVBA :: Av. de l'Arme 68 B4 :: BE-1040 BELGIUM

Email: info@gentaur.com
 

International 

+32 (0) 16 58 90 45

+32 (0) 16 50 90 45

France

01 43 25 01 50

01 43 25 01 60

Italy

02 36 00 65 93

02 36 00 65 94

Germany

0241 6085 13140

0241 6085 33033

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Copyright 2005 Gentaur BVBA
Last modified: feb-07