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
REAGENTS
COTININE Direct ELISA Kit Contents
Component
96 test Kit
96 well Micro-plate
1
Cotinine Conj.
15 mL
Neg Serum Std
1 mL
Cotinine 5 ng/mL
1 mL
Cotinine 10 ng/mL
1 mL
Cotinine 25 ng/mL
1 mL
Cotinine 50 ng/mL
1 mL
Cotinine 100 ng/mL
1 mL
Neg Urine Std
2 mL
Cotinine Pos Std
2 mL
TMB Substrate
30 ml
Stop Reagent
25 ml
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
azide preservatives.
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 required).
Manual or electronic micropipets (single channel or multichannel) or automated pipetting stations.
Refrigerator (for kit storage).
Interval Timer.
Wash bottle or Plate Washer.
Microplate reader capable of reading at 450 nm. And 650 nm.
Precautions
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 reliable delivery.
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 the substrate.
SPECIMEN COLLECTION
Precautions.
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.
Additives.
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.