LSY-10062 TheCiprofloxacin ELISA Test Kit 1ppb antibiotic residue detection

TheCiprofloxacin ELISA Kit

Catalog No. LSY-10062

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of theCiprofloxacin in samples. The coupling antigens are pre-coated on the micro-well stripes. TheCiprofloxacin in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-theCiprofloxacin antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with theCiprofloxacin in the sample. This value is compared to the standard curve and theCiprofloxacin concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 1 ppb

Incubation Temperature: 25℃

Incubation Time: 30min—15min

Detection limit: about 1ppb

Cross-reaction rate:

TheCiprofloxacin (CIF) 100%

Recovery rate

Tissue,Egg, Serum 80±15%

Honey 75±15%

3. Components

• Micro-well strips: 12 strips with 8 removable wells each
• 6× standard solution (1 mL each): 0ppb, 1ppb, 3ppb, 9ppb, 27ppb and 81ppb
• Enzyme conjugate (7ml) red cap
• Antibody working solution (7 mL) blue cap
• Substrate A solution (7 mL) white cap
• Substrate B solution (7 mL) black cap
• Stop solution (7 mL) yellow cap
• 20× concentrated washing buffer (40 mL) white cap
• 2× concentrated redissolving solution (50 mL) transparent cap

4. Materials required but not provided

• Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g)
• Micropipettors: single-channel 20-200 µL, 100-1000 µL, and multi-channel 250 µL;
• Reagents: HCl, Methylene chloride, Acetonitrile (CH₃CN), N-hexane, Na₂HPO₄·12H₂O, NaH₂PO₄·2H₂O, Heparin sodium

5. Sample pre-treatment

Instructions (The following points must be dealt with before the pre-treatment)

1) This test kit can detect tissue sample: animal tissue, poultry, aquatic. Eg: Chicken, duck, bovine, rabbit, fish, shrimp etc.

• Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;
• Before the experiment, each experimental utensil must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

• 0.1 M HCl: 860µl HCI (36%) + deionized water 100 mL.
• Acetonitrile (CH₃CN) -Methylene chloride mixing solution

V_acetonitrile-V_{methylene chloride} = 1 : 4

• pH7.2 0.02M PB buffer: dissolve 5.16 g Na₂HPO₄·12H₂O + 0.87 g NaH₂PO₄·2H₂O in the deionized water to 1 L.
• Acetonitrile (CH₃CN)- Methylene chloride- 0.1 M HCl mixing solution

100ml Acetonitrile (CH₃CN) -Methylene chloride mixing solution (V_acetonitrile-V_{methylene chloride} = 4 : 1), add 5ml 0.1 M HCl solution.

• The 2× concentrated redissolving solution is diluted with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water), used for the sample redissolving.

5.1 Tissues (chicken/liver, pork/liver, fish, shrimp etc.),egg

1) Weigh 2.0 ± 0.05 g of the homogenized tissue sample into 50 ml centrifuge tube

2) Add 8 ml of the Acetonitrile (CH₃CN) -Methylene chloride mixing solution, shake for 5 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min

3) Take 4 ml the clear organic phase (upper layer) into a dry tube, blow to dry with nitrogen or air completely by rotary evaporation at 56 ℃

• Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 30 seconds; centrifuge at above 4000 r/min at 15℃ for 5 min.
• Remove the upper layer, take 50µl lower layer solution for further analysis.

Fold of dilution of the sample: 1

5.2 Serum

1) Use centrifuge tube with heparin sodium (20-30 unit/ml blood) to collect chicken blood sample (Suggestion: blood collection syringes are recommended rinsing with heparin).Place the blood sample in the room temperature for 1 hour. After obtain plasma, centrifuge at above 4000r/min at 15 ℃ for 10 min, take out 1 ml plasma.

2) Add CH₃CN (without water) 4 ml , mix up-and-down thoroughly for 5 min, centrifuge at above 4000r/min at 15 ℃ for 10 min.

3) Move the clear supernatant (upper layer) to another centrifuge tube, add 2ml 0.02M PB buffer, mix evenly.

4) Add 5 ml Methylene chloride, mix evenly for 5 min, centrifuge at above 4000r/min at

15 ℃ for 10 min, remove the upper layer, take the lower organic phase to dry bottle(clear without impurities), blow to dry with nitrogen or air completely by rotary evaporation at 50 ℃

5) Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 30 seconds; centrifuge at above 4000 r/min at 15℃ for 5 min.

6) Absorb out lightly the upper and middle layer white impurities, take lower phase 100µl, add 100µl diluted redissolving solution, mix for 30s.

7) Take 50µl solution for further analysis.

Fold of dilution of the sample: 2

5.3 Honey

• Weigh 2.0±0.05g honey sample into 50ml centrifuge tube, add 8 ml Acetonitrile (CH₃CN)- Methylene chloride- 0.1 M HCl mixing solution, shake fully for 3min, centrifuge at above 4000 r/min at 15 ℃ for 10 min.
• Take 2ml the supernatant (upper layer), blow to dry with nitrogen or air at 56 ℃.
• Add 1 mL the diluted redissolving solution, shake fully for 1min.
• Take 50 µL for further analysis

Fold of dilution of the sample: 2