Search，Order，Antibody,ELISA Kit & Protein

1. SUMMARY

Ampicillin penicillin (AM) Ampicillin, is widely used for livestock diseases have antibiotics, control and treatment played an important role. But because ampicillin penicillin can cause allergic reactions and bacteria resistant and other reasons, Europe andChinaand other countries has successively requirements set limit to use. Because current use of testing analysis sample pretreatment and determination of operating trival and cost is higher, make application is restricted.This kit is the application of the new generation technology r&d ELISA drug residue detection products, operation time only 1.5 hours, can minimize errors and working intensity.

2. PRINCIPLE

This kit by indirect competitive ELISA method, the microporous bar pre bag was coupling antigen, sample of residue ampicillin penicillin will and microporous bar represents the bag was coupling antigen competition ampicillin penicillin antibody, join enzyme standard, with TMB after two resistance of substrate colour-display, sample absorb light value and contained residues ampicillin penicillin content of a negative correlation with standard curve, comparative multiplying by its corresponding diluted times can draw the corresponding residue ampicillin penicillin content. Applicable scope can qualitative and quantitative detection of animal tissue (muscles and liver, etc.), honey as well as in their sample ampicillin penicillin footprint.

3. CROSS REACTION RATE

Ampicillin penicillin............ 100%

Penicillin............ 52 %

Penicillin V............ < 20%

Crossing with other penicillin drugs are less than 2%

4.SELF-PROVIDED EQUIPMENT AND REAGENTS IN USING UNIT

 (1)S SELF-PROVIDED EQUIPMENT

—Microporous board microplate 450nm / 630nm

—Rotating evaporation instrument/nitrogen blow dry device

—Homogenizer can

—Oscillator

—Vortex instrument

—Centrifuge

—Balance: feeling quantity 0.01 g

—Scale pipette: 10ml

—Wash our ears ball

—RongLiangPing: 100ml canister, 500ml

—Polystyrene centrifugal tube: 5ml, 50ml

—Glass tube: 10ml

—Trace remove liquid device: single-channel 20ml ~ ~ 1000ml 200ml, 100ml canister，Multi-channel 250ml

（2）REAGENTS IN USING UNIT

—Sodium hydroxide (analysis pure)

—Acetonitrile (analysis pure)

—Are hexane (analysis pure)

—Sulfuric acid (honey specific)

—Strong hydrochloric acid (honey specific)

—Deionized water

5. MATERIALS AND REAGENTS PROVIDED

(1)96-well microtiter plates ×1piece （coated with Coupling antigens）

(2)Standard solution×6bottle：（1ml/bottle）0 ng/ml，0.2 ng/ml，0.6 ng/ml，1.8 ng/ml，5.4 ng/ml，16.2 ng/ml

(3)High concentration of standard：（1ml/bottle）100 ng/ml

(4) ELIAS secondary antibody   12ml ………………………… Red hat

(5)Antibody working solution    7ml………………………Red hat

(6) Substrate solution A         7ml …………………………White hat

(7)Substrate solution B         7ml………………………Red hat

(8) Stop solution               7ml………………………… Yellow hat

(9)20×concentrated washing solution  40ml ………………………… Hyaline hat

(10)20×concentrated washing solution    40ml ………………………… Hyaline hat

(11) 2×sample dilution          50ml …………………………Blue hat

6.SOLUTION OF PREPARATION

With liquid 1: Washing liquid,Use deionized water will be 20 "x" volume concentration by washing the ratio of 20 dilution (1 + detergent x enrichment) for 19 of deionized water enzyme panels of catharsis, washing liquid in 4 ℃ environment can save a month.

With liquid 2:0.1 M sodium hydroxide solution (milk method c, organization, honey method),Says take 0.4 g sodium hydroxide add deionized water 100ml canister to dissolve.

With liquid 3: Acetonitrile -0.1 M sodium hydroxide solution (organization) quantity with 84ml extraction take 16ml 0.1 M acetonitrile and mixed all sodium hydroxide Well.

With liquid 4: Acidification acetonitrile solution (honey specific)，Quantity take 100ml canister acetonitrile join150ml 2M sulfuric acid mixture (kit for the termination of liquid concentration in 2M/L of sulfuric acid).

With liquid 5:1-m hydrochloric acid solution (honey specific)，Quantity took 8.3 ml strong hydrochloric acid join deionized water the capacity to 100ml canister.

With liquid 6:1-m naoh water solution (honey specific)，Says take 4.0 g sodium hydroxide add deionized water the capacity to 100ml canister.

With liquid 7: Washing liquid，Use deionized water will be 20 "x" volume concentration by washing the ratio of 20 dilution (1 + detergent x enrichment) for 19 of deionized water enzyme panels of catharsis, washing liquid in 4 ℃ environment can save a month.

7. SAMPLE PRETREATMENT STEPS

Sample processing notes before

Handle any samples, must note:

(a) Experiments must use one-off suction head, in the use of different reagent to replace suction head.

(b) Before tests must check whether various experimental instruments is clean, must use clean experimental apparatus, to avoid pollution interference experiment results.

(c) Non-operated samples should be cryopreservation.

(d) Handle good sample shall be immediately used for analysis.

(a) Organization (chickens, ducks, pork/pork liver, shrimp, fish) pre-treatment methods

(1)HOMOGENIZER TISSUE SAMPLES

—Take 2.0 + 0.05 g homogeneous content to 50ml polystyrene centrifugal tube, join 8ml acetonitrile -0.1 M sodium hydroxide solution (see with liquid 3), using vortex instrument vortex move 2min, reoccupy oscillator oscillation, 3000g 10min at room temperature (more than 20-25 ℃) centrifugal 10min,

—Removed 1ml supernatant fluid to 10ml clean glass tube, in 50 ~ 60 ℃ water-bath blow dry nitrogen flow,

—To join 1ml are hexane dissolve dry residual, add 1ml complex dissolve working liquid (see with liquid 1) fully blending, and 3000g above, room temperature (20-25 ℃) centrifugal 5min;

—Remove upper are hexane phase, and the lower water Moluccas complex soluble working liquid press and the volume ratio for dilution (50ml sample extract + 200ml complex dissolve working liquid);

—Take 50 ml used for analysis.

(2) HONEY

—Take 4.0 + 0.05 g honey sample to 50ml polystyrene centrifugal tube, join 0.5 ml 1-m naoh water solution (see with liquid 6), use oscillator oscillation mixed 20min quiet place,

—Join 0.5 ml 1-m hydrochloric acid solution (see with liquid 5), use oscillator oscillation blending (pH should be in 3 or so, if not in this range please use hydrochloric acid or sodium hydroxide transferred to accurate), then add 7ml acidification acetonitrile (controlled) (see pH4.0 with liquid 4), with the oscillator, 3000g above 10min fully oscillation, room temperature (20-25 ℃) centrifugal 10min,

—Take supernatant fluid to 3ml 10ml clean glass tube, in 50 ~ 60 ℃ water-bath blow dry nitrogen flow,1ml after dissolution -- addition of working liquid (see with liquid 1), using vortex instrument 2min vortex move,

—Take 50 l used for analysis.

8.DETECTING STEPS

（1）DETERMINATION NOTES BEFORE THE EXPERIMENT

1）Rise the temperature of all kit and the need to room temperature before using(20-25 ℃).

2）Put back all reagent 2-8 ℃immediately after use.

3）The analysis of ELISA reproducibility depends largely on the consistency of the washing board,

the correct operation of washing board is the point in the determination program of ELISA.

4）During all thermostatic incubation process, it should avoid light irradiation, cover plate with

sealed microporous membrane.

（2）OPERATION STEPS

1）Removed reagents from refrigeration environment, bring them to room temperature (20-25 ℃)

balance 30min above, note each liquid reagent is required to shake well before use.

2）Remove the required number of microporous board, put back unneeded microporous into

self-appointed bag, and keep in 2-8 ℃.

3）Washing liquid also needs to room temperature before use.

4）Numbering: numbering the sample and standard substance corresponding microporous the

sequential Numbers, each sample and standard parallel make 2 holes and record the standard holes

and sample hole position.

5）Adding standard /sample: add 50ml standard/samples to corresponding microporous, then add

the antibody working liquid 50ml/hole, gently oscillation blending, and cover with membrane

rearmounted at 25 ℃ avoiding light environment to react 30min.

6）Washing board: carefully open the liquid membrane, dry the hole, fully washing 4-5 times with

scouring working liquid (see with liquid 1) 250ml/hole, , each time interval 10s, pat dry with an

absorbent (after pat dry, pierce the unclear bubble with unused spear).

7）Add HRP secondary antibody: Add HRP secondary antibody100ml/hole, gently oscillation

blending, and cover with cover membrane at 25 ℃ avoiding light environment reaction 30min,

repeat washing board of step 6.

8）Show color: join substrate liquid A liquid 50ml/hole, add substrate liquid B liquid 50ml/hole,

gently oscillation blending, and cover with membrane rearmounted at 25 ℃ avoiding light

environment and let react 15min (see note 8).

9）Determination: join terminated liquid 50 ml/hole, gently oscillation blending, and set microplate

in 450nm place (suggest using dual wavelength 450/630nm to detect, please finish reading data in

5min), detect OD value of each hole. (if there is no terminated liquid, it can undertake judging by

eye-measurement).

9.RESULT

Result determination have two kinds of methods, roughly judgement usable 1 method, and quantitative determination by the first two methods. Note sample absorb light value and ampicillin penicillin content of a negative correlation.

1.Using the sample of average absorbance of the value and the standard comparison can obtain its concentration range (PPB). Hypothesis sample 1 absorbance of the value of 0.190, sample 2 absorbance of the value of 0.742, standard fluid absorbency value is respectively: for 0ppb 1.802; For 1.428; PPB 0.2 - For 1.011 0.6 PPB, For 0.560; 1.8 PPB For 0.257; PPB 5.4, 16.2 PPB for 0.110. Then sample 1 concentration range is 5.4 PPB - 16.2 PPB multiplying by the corresponding diluted times can draw in their sample ampicillin penicillin residual concentration range, Sample 2 concentration range is 0.6 PPB - 1.8 PPB multiplying by the corresponding diluted times can draw in their sample ampicillin penicillin residual concentration range.