Trimethoprim ELISA Test Kit

Catalog No. LSY-10065

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Trimethoprim in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Trimethoprim in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Trimethoprim antibody. 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 the Trimethoprim in it. This value is compared to the standard curve and the Trimethoprim concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.5 ppb

Incubation Temperature: 25℃

Incubation Time: 30min—15min

Detection limit:  

Tissue 5ppb

Cross-reaction rate:

Trimethoprim 100%

Sulfamerazine  ＜0.1%

Sulfaquinoxaline  <0.1%

Sulfamethazine  ＜0.1%

Sulfamonomethoxine  <0.1%

Sulfamethoxazole  <0.1%

Sulfathiazole  <0.1%

Sulfamethoxypyridazine  <0.1%

Sulfaguanidine  <0.1%

Sulfadimethoxine  <0.1%

Sulfadiazine   <0.1%

Sulfameter   <0.1%

Recovery rate:

Tissue 60%～120%

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1mL each)	0ppb	0.5ppb
		1.5ppb	4.5ppb
		13.5ppb	40.5ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A	7ml	white cap
6	SubstrateB	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	15ml	white cap
9	20×sample extractA	15ml	yellow cap
10	2×sample extractB	50ml*2	transparent cap
11	10× sample diluent	10ml	black cap

4. Materials required but not provided

1) Equipments: microplate reader, homogenizer, oscillator, centrifuge, balance (a sensibility reciprocal of 0.01 g), graduated pipette, incubator.

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

3) Reagents: NaOH.

 

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

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 equipment 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) Sample extract A

1 part of 20× sample extract A + 19 parts of deionized water

2) Sample extract B

1 part of 2× sample extract B + 1 part of deionized water

3)1M NaOH solution

Weigh 4g NaOH, add deionized water to 100ml

4)Sample diluent

1 part of 10× sample diluent + 9 parts of deionized water

5.1 Tissue  

1) Take 2± 0.05 g of the homogenized tissue sample into 50 mL centrifuge tube, add 3mL diluted Sample extract A, shake for 3min;

2) Then add 600ul 1M NaOH solution and 2.4ml diluted Sample extract B, shake for 3min; centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 5 minutes.

3) Take 200ul up-layer clear liquid, add 300ul diluted Sample diluent, mix it evenly;

4) Take 50ul above mixed solution for analysis.

Fold of dilution of the sample:10

 

6.ELISA procedures

 Instructions

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

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.

 Operation procedures

1. Take out all the necessary reagents from the kit and place at the room temperature (20 to 25 ℃) for at least 30 minutes. Note that each liquid 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 ℃, not frozen.

3. Washing buffer preparation: dilute 15 mL of the 20× concentrated washing buffer with the deionized water at 1:19 (1 part 20× concentrated washing buffer + 19 parts deionized water ). Or prepare washing buffer 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. Add 50µL of the sample or standard solution to separate duplicate wells, and add 50ul Enzyme conjugate then 50 µL of the antibody solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25℃ for30 minutes.

6. Pour liquid out of microwell, add 250 µL/well of washing buffer for 15-30 seconds, repeat four to five times, then flap to dry (if there are the bubbles after flapping, cut them with the clean tips).

7. Coloration: add 50 µL of the substrate A and then 50 µL of the substrate B into each well. Mix gently by shaking the plate manually, andincubate at25 ℃ for15 minutes at dark for coloration.

8. Determination: add 50 µL of the stop solution into each well. Mix gently by shaking the plate manually. 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 minutes).

 

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 of Trimethoprim in the sample.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from the comparison the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sample Ⅰ is 0.3, and that of the sample Ⅱ is 1.0, while those of the standard solutions are as the followings: 2.243 for 0ppb, 1.816 for 0.5ppb, 1.415 for 1.5ppb, 0.74 for 4.5ppb, 0.313 for 13.5ppb and 0.155 for 40.5ppb, accordingly the concentration range of the sampleⅠis 13.5 to 40.5ppb, and that of the sample Ⅱ is 1.5 to 4.5ppb.

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 0 ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Trimethoprim 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, thus finally obtaining Trimethoprim 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.

3. Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility.

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

5. 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.

6. 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.

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 indicates its degeneration.

8. The optimum reaction temperature is 25 ℃, 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: 12 months; date of production is on the 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.