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0032 (0)16 41 44 07
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0032 (0)16 41 44 07
COTININE Direct Elisa
Catalog Number: 130BQ086D (96 wells)
EXPLANATION OF THE TEST
This Cotinine Direct ELISA Kit is a specific and sensitive in-vitro test to
detect the presence of cotinine in serum and urine. Exposure to tobacco smoke
can be detected by measuring nicotine and its metabolites. Nicotine has a short
half life and is not used as a marker for tobacco smoke exposure. Cotinine due
to its longer half life has been used in research as a reliable marker for
smoking status and smoking cessation studies.
PRINCIPLES OF THE PROCEDURE
This Cotinine Direct ELISA Kit is based upon the competitive binding to antibody
of enzyme labeled antigen and unlabeled antigen, in proportion to their
concentration in the reaction mixture.
A 10 μl. aliquot of a diluted unknown specimen is incubated with a 100 μl.
dilution of enzyme (Horseradish peroxidase) labeled Cotinine derivative in
micro-plate wells, coated with fixed amounts of oriented high affinity purified
polyclonal antibody. The wells are washed thoroughly and a chromogenic substrate
added. The color produced is stopped using a dilute acid stop solution and the
wells read at 450 nm. The intensity of the color developed is inversely
proportional to the concentration of drug in the sample. The technique is
sensitive to 1 ng/ml
COTININE Direct ELISA Kit Contents
96 test Kit
96 well Micro-plate
Neg Serum Std
Cotinine 5 ng/mL
Cotinine 10 ng/mL
Cotinine 25 ng/mL
Cotinine 50 ng/mL
Cotinine 100 ng/mL
Neg Urine Std
Cotinine Pos Std
96 well micro-plate. The micro-plate is coated with polyclonal antibody to
Cotinine via a spacer chain to provide optimally oriented binding sites. The
plates are sealed in a moisture and air barrier pouch with a dessicant.
Cotinine-Enzyme Conjugate The conjugate solution contains a Cotinine derivative
labeled with horseradish peroxidase in a buffered, protein solution with
stabilizers, pH 7.6 containing non
Negative Serum Standard. This bottle contains drug free rabbit serum containing
azide free preservatives.
Serum Cotinine Standards. These bottles contain 5, 10, 25, 50 and 100 ng/mL of
Cotinine dissolved in a rabbit serum non azide preservative.
Negative Urine Standard. This bottle contains drug free synthetic urine matrix
containing azide free preservatives Urine Cotinine Positive Standard. This
bottle contains 500 ng/mL of cotinine in a synthetic urine matrix containing
azide free preservatives.
TMB chromogenic substrate. The color reagent contains 3,3’,5,5’
tetramethylbenzidine and urea peroxidase in buffer.
Stop Reagent This contains 1 N hydrochloric acid.
Materials and Equipment
Materials and equipment required but not supplied with this Cotinine Direct
ELISA Kit are itemized below
12x75 mm Disposable Glass or Plastic Culture Tubes to predilute samples (if
Manual or electronic micropipets (single channel or multichannel) or automated
Refrigerator (for kit storage).
Wash bottle or Plate Washer.
Microplate reader capable of reading at 450 nm. And 650 nm.
1. Not for Internal or External Use in Humans or Animals.
2. There should be no eating or drinking within work area.
3. Always wear gloves and a protective lab coat.
4. No pipetting should be done by mouth. Handle all specimens and reagents as
potentially infectious and biohazardous.
5. Do not add sodium azide to samples as preservative.
6. Do not use external controls containing sodium azide.
7. Use disposable pipet tips to avoid contaminating chromogenic
substrate reagent. Discard reagent if it turns blue.
8. Do not pour chromogenic substrate back into container after use.
9. Do not freeze reagents.
10. Do not mix reagents from different kit lot numbers.
11. Keep reagents out of direct sunlight.
12. Handle stop reagent with care, since it is corrosive.
13. Bring all reagents to room temperature.
14. Viscous forensic samples should always be diluted in phosphate buffered
saline or distilled water prior to pippetting.
15. Ensure the bag containing the micro-plate strips and dessicant is well
sealed if only a few strips are used.
General. Precise pipetting is the essence of successful immunoassay. It is
critical to pipet right at the center and bottom of each well to ensure good
replicates and coefficents of variation Micropipets supplied by "Eppendorf" or
"SMI" with disposable tips are excellent when used carefully according to
instructions to insure the necessary accuracy. New automatic dispensers improve
Storage. The expiration date of the kit is stated on the label. The kit can be
expected to perform satisfactorily until the expiration date if stored in the
refrigerator at 2 – 4 °C.
Indications of Deterioration. A drop of greater than 50% in the A0
(zero-standard absorbance reading) for a constant incubation time indicates
deterioration of the antibody plate, enzyme conjugate or chromogenic substrate.
A significant shift of the standard curve to the right would result from
deterioration of the standards. Development of blue color in the chromogenic
substrate without the addition of enzyme conjugate indicates contamination of
This Cotinine Direct ELISA Kit is to be used with human urine or serum. This
assay has not tested for all possible applications. Cutoff criteria are
important in deciding the sample dilution.
Specimens to which sodium azide has been added affect the assay.
DETAILS OF THE PROCEDURE.
All reagents must be brought to room temperature (20-25 °C) before use.
The procedure as described below may be followed in sequence using manual
pipettes. Alternatively all reagents may be added using an automated pipettor.
Use urine calibrators for urine and serum calibrators for serum. Depending on
the cutoffs a sample dilution may be required for urine applications.