Cefalexin ELISA Test Kit

Catalog No. LSY-10067

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection ofCefalexin insamples. The coupling antigensare pre-coated on the micro-well stripes.TheCefalexin in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Cefalexin 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 theCefalexin in the sample. This value is compared to the standard curve and theCefalexin concentration is subsequently obtained.

2. Technical specifications

Sensitivity:0.3 ppb

Incubation Temperature: 25℃

Incubation Time: 30min—15min

Detection limit: 

Tissue  10ppb

Egg 15ppb

Liquid milk 5ppb

Milk powder 24ppb

Cross-reaction rate:

Cefalexin 100%

Recovery rate

Tissue100%±30%

Egg, liquid milk, milk powder 90%±30%

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1mL each)	0ppb	0.3ppb
		0.9ppb	2.7ppb
		8.1ppb	24.3ppb
3	High concentration standard	1ml	1 ppm
4	Enzyme conjugate	7ml	red cap
5	Antibody working solution	7ml	blue cap
6	Substrate A solution	7ml	white cap
7	SubstrateBsolution	7ml	black cap
8	Stop solution	7ml	yellow cap
9	20× extracting buffer A	15ml	yellow cap
10	2× extracting buffer B	50ml*2	transparent cap
11	20× concentrated washing buffer	15ml	white cap
12	10× concentrated redissolving solution	10ml	black cap

4. Materials required but not provided

1) Equipments:microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g), incubator.

2) Micropipettors: single-channel 20-200 µL,100-1000 µL, and multi-channel 30～300 µl ;

3) Reagents: Methanol, NaOH

5. Sample pre-treatment

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

1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2) 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:

1) 1M NaOH:Take 4.0g NaOH, add deionized waterto100mL, mix it evenly.

2) 10%Methanol: 1 part ofMethanol + 9 parts ofdeionized water, mix it evenly.

3) The10× concentrated redissolving solution is diluted with deionized water at 1:9 (1part of 10x concentrated redissolving solution +9 parts of deionized water), mix it evenly.

4) Extracting buffer A:1 part of20× extracting buffer A + 19 parts ofdeionized water, mix it evenly.

5) Extracting buffer B:1 part of 2× extracting buffer B + 1 part ofdeionized water, mix it evenly.

5.1Tissue

1) Weigh2.0 ± 0.05 g of the homogenizedtissuesample into 50 ml centrifuge tube, add 3mlExtracting buffer A, vortex for 3min;

2)  Then add 600µl 1M NaOH and 2.4mlExtracting buffer B,vortex for3 min,centrifuge at above 4000 r/min atroom temperature for5 min;

3)  Take100µl up-layer liquid, add 400µl dilutedredissolving solution, mix it evenly;

4)  Take 50µl solution for further analysis.

Fold of dilution of the sample: 20

5.2Egg

1)  Weigh 1±0.05g egg sample into 10mlcentrifuge tube, add 5ml 10% Methanol, vortex for 3min,centrifuge at above 4000 r/min atroom temperature for5 min; take 100ul up-layer liquid, add 900µl diluted redissolving solution, shake for 30s;

2)  Take 50µl solution for further analysis.

Fold of dilution of the sample:50

5.3 Liquid milk

1. Take100µl liquid milk into2mlcentrifuge tube, add 900µl diluted redissolving solution, vortex for 30s,centrifuge at above 4000 r/min atroom temperature for5 min;

2. Take50 µLup-layer liquidfor further analysis

Fold of dilution of the sample:10  

5.4 Milk powder

1) Weigh1.0 ± 0.05 g of themilk powder sample into10 ml centrifuge tube, add 7mldeionized water, vortex for 3min,centrifuge at above 4000 r/min atroom temperature for5 min;

2) Take100µl up-layer liquid into 2mlcentrifuge tube, add 900µl dilutedredissolving solution, vortex for 30s,centrifuge at above 4000 r/min atroom temperature for5 min; ;

4)  Take 50µl up-layer liquid for further analysis.

Fold of dilution of the sample: 80

6. ELISA procedures

6.1 Instructions

1. Bring all reagents and micro-well strips to the room temperature (20-25℃) before use;

2. Return all reagents to 2-8℃ immediately after use;

3. The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in the procedures of ELISA;

4. For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.

6.2 Operation procedures

1. Take out all the necessary reagents and place at the room temperature (20-25℃) for at least 30min. Note that each reagent must be shaken to mix evenly before use;

2. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2- 8℃;

3. Solution preparation: dilute15 mL ofthe 20× concentrated washing buffer with the deionized water at 1:19 ( 1 part of20× concentrated washing buffer + 19 parts of deionized water), or prepare as quantity needed;

4. Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions;

5. Add50 µL of the sample or standard solution to separate duplicate wells, add 50 µL of enzyme conjugate, then add50 µL of the antibody working solution into each well. Mix by shaking gently, seal the microplate with the cover membrane, andincubate at 25℃ for30min;

6. Wash the microplate with the washing buffer at 250 µL/well for four to five times.;soak the well with the washing buffer for 15-30 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips);

7. Coloration: add 50 µL of the substrate A solution and 50 µL of the substrate B solution into each well. Mix by shaking gently, and incubate at 25℃ for15 min in the dark for coloration;

8. Determination: add 50 µL of stop solution into each well. Mix by shaking gently. Set the wavelength of the microplate reader at 450 nm to determine the OD value. (recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min) .

7. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content ofCefalexin in the sample.

7.1 Qualitative determination

The concentration range (ng/mL) obtained from the comparison the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is1.0, the OD value of standard solutions is:2.243 for 0ppb, 1.816 for0.3ppb, 1.415 for0.9ppb, 0.74 for2.7ppb, 0.313 for8.1ppb ,0.155for24.3ppb, accordingly the concentration range of the sampleⅠ is8.1ppb～24.3ppb, and that of the sampleⅡ is0.9ppb～2.7ppb. (multiplied by the corresponding dilution fold)

7.2 Quantitative determination

The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is,

B—the average (double wells) OD value of the sample or the standard solution

B0—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of theCefalexin standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, finally obtaining theCefalexin concentration in the sample.

Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software)

8. Precautions

1. The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value.

2. Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility; So continue to next step immediately after washing.

3. Mix evenly, otherwise there will be the undesirable reproducibility.

4. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.

5. Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.

6. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light.

7. Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of the 0 standard solution of less than 0.5 (A450 nm< 0.5 ) indicates its degeneration.

8.  The optimum reaction temperature is25℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

Expiry date: 1 year; date of production is on box.

Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.