CN214703651U - Fluorescence immunochromatography test strip and fluorescence immunochromatography test card - Google Patents

Abstract

The utility model relates to a fluorescence immunochromatographic test strip and fluorescence immunochromatographic test card, fluorescence immunochromatographic test strip include the bottom plate and locate sample pad, combination pad, reaction film and the pad that absorbs water on the bottom plate, the sample pad the combination pad the reaction film with the pad that absorbs water overlaps in proper order, be equipped with the detection line on the reaction film, the reaction film is close to the one end of combination pad with distance between the detection line is 1.35cm ~ 1.85cm, the parcel is had the capture antibody on the detection line, contain the mark antibody on the combination pad. The fluorescence immunochromatographic test strip optimizes the position of a detection line in order to improve the linear range of the test strip, and is beneficial to increasing the linear range of the test strip.

Description

Fluorescence immunochromatography test strip and fluorescence immunochromatography test card

Technical Field

The utility model relates to an immunochromatography technical field especially relates to a fluorescence immunochromatography test paper strip and fluorescence immunochromatography test card.

Background

Serum amyloid a (saa) is a major Acute Phase Protein (APP) found in many species, including humans, dogs, cats and horses. Like CRP, SAA can serve as a sensitive marker of systemic inflammation. SAA is highly conserved in vertebrates. Human SAA contains 104 amino acids, whereas canine, feline, and equine SAA contains 111 amino acids, and equine SAA contains 110 amino acids.

In veterinary medicine in horses, SAA has proven to be a major APP, and after trauma, infection or inflammation has occurred, the SAA content in the serum of horses has rapidly increased to more than 1000-fold and after recovery or upon effective treatment has immediately returned to normal levels, so that measuring the concentration of SAA in the blood can be used to detect the presence of inflammation, and as a prognostic marker for various diseases, such as equine abdominal pain and sepsis, etc., as well as for diagnosing subclinical inflammation, monitoring the treatment of inflammation and infection in animals and monitoring the progress of surgical procedures in patients. With the increasing standardization of the domestic horse veterinary diagnosis and treatment industry, horse SAA detection becomes a powerful method for horse disease diagnosis. The immunochromatography technology is an analysis method combining the immunity technology and the chromatography technology, which is developed at the end of the 20 th century, and the method has the characteristics of simple and quick operation and the like, but the linear range of a general fluorescence immunochromatography test strip is narrow, and the requirement of detecting the horse serum amyloid A cannot be met.

SUMMERY OF THE UTILITY MODEL

Therefore, there is a need for a fluorescence immunochromatographic test strip with a wide linear range.

The utility model provides a fluorescence immunochromatographic test strip, includes the bottom plate and locates sample pad, combination pad, reaction membrane and the pad that absorbs water on the bottom plate, the sample pad, the combination pad, the reaction membrane with the pad that absorbs water overlap joint in proper order, be equipped with the detection line on the reaction membrane, the reaction membrane be close to the one end of combination pad with the distance between the detection line is 1.35cm ~ 1.85cm, the detection line is gone up the parcel and is had the capture antibody, contain the mark antibody on the combination pad.

The utility model discloses a fluorescence immunochromatographic test strip has optimized the position of detection line for improving its linear range, makes the reaction membrane be close to the one end of combination pad and the distance between the detection line be 1.35cm ~ 1.85cm, is favorable to increasing the linear range of test strip. The principle is that when the sample is specifically combined with the labeled antibody on the binding pad, an antigen-antibody fluorescent complex is formed, and within a certain range, when the detection line is close to the binding pad end, the reaction time of the sample and the labeled antibody is shortened, the number of the labeled antibodies which can be captured is reduced, the signal is weakened, and the increase of the linear range is facilitated; when the detection line is far away from the end of the binding pad, the reaction time of the sample and the labeled antibody is increased, the amount of the labeled antibody which can be captured is increased, the signal is strengthened, and the increase of the linear range is not facilitated. Proved by experiments, the fluorescence immunochromatographic test strip of the utility model can reliably measure the SAA concentration in a very wide concentration range (8 mg/L-1000 mg/L), thereby distinguishing horses with different inflammatory states, being used for monitoring the change of inflammatory activity and having very high applicability in equine species.

In one embodiment, the fluorescence immunochromatographic test strip further comprises a blocking pad arranged on the bottom plate, the sample pad, the blocking pad, the binding pad, the reaction membrane and the water absorption pad are sequentially overlapped, and the blocking pad contains a blocking agent.

In one embodiment, the overlapping length of the blocking pad and the combining pad is 1.5 mm-3 mm.

In one embodiment, the length of the blocking pad along the extending direction of the fluorescence immunochromatographic test strip is 0.8cm to 1.2 cm.

In one embodiment, the blocking pad is a fiberglass pad or a polyester film pad.

In one embodiment, the reaction membrane is further provided with a quality control line, the detection line is closer to the water absorption pad than the quality control line, and the quality control line is coated with a quality control antibody.

In one embodiment, the distance between the quality control line and the detection line is 3 mm-5 mm.

In one embodiment, the length of the binding pad along the extending direction of the fluorescence immunochromatographic test strip is 1 cm-2 cm, and the length of the water absorption pad along the extending direction of the fluorescence immunochromatographic test strip is 2 cm-3 cm.

In one embodiment, the base plate is a PVC plate having an adhesive on a surface thereof.

The utility model also provides a fluorescence immunochromatography detects card, including above-mentioned fluorescence immunochromatography test paper strip and cutting ferrule, fluorescence immunochromatography test paper strip is located in the cutting ferrule, one of them surface of cutting ferrule has sample application window and display window, the sample pad with the sample application window is relative, the detection line with the display window is relative.

Drawings

Fig. 1 is a schematic structural diagram of a fluorescence immunochromatographic test strip in one embodiment;

FIG. 2 is a graph showing a comparison of the results of detection by the detection kit of the embodiment and the conventional detection kit;

FIG. 3 is a schematic diagram of different positions of the detection lines in the embodiment;

FIG. 4 is a diagram illustrating the detection results of the detection lines at different positions in the embodiment;

FIG. 5 is an equation and fitted linear regression curve established in the examples with the mean value of T/C as ordinate and the equine SAA recombinant antigen reference as abscissa.

Detailed Description

In order to facilitate understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention will be described. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

As shown in fig. 1, the utility model discloses a fluorescence immunochromatographic test strip 100 of embodiment, including bottom plate 10 and locate sample pad 20 on bottom plate 10, combination pad 30, reaction membrane 40 and absorb water pad 50, sample pad 20, combination pad 30, reaction membrane 40 and absorb water pad 50 overlap joint in proper order, are equipped with detection line 42 on the reaction membrane 40, and the one end that reaction membrane 40 is close to combination pad 30 is 1.35cm ~ 1.85cm with the distance between detection line 42, and the detection line 42 is gone up the parcel and is had the capture antibody, contains the mark antibody on the combination pad 30.

Due to species specificity, horses appear unique in their ability to produce large amounts of SAA in response to inflammatory stimuli, ranging from 0mg/l in healthy individuals to several thousand mg/l in individuals with severe inflammation. The sustained low concentrations and extreme extent of the inflammatory response found in healthy horses make SAA a particularly useful marker of inflammation in horses. When the analyte is in excess and all antibodies in the assay are fully saturated, a prozone effect (also known as the hook effect) may occur. This requires multiple manual dilutions of the sample, which increases reagent and labor costs and may result in an erroneously low concentration output, affecting the reliability of the results, and is therefore a highly undesirable feature. In order to be able to better measure the severity/extent of inflammation and monitor changes in inflammatory activity in critically ill horses, an ideal SAA test must be able to be reliably measured over an extremely wide concentration range without the need for multiple manual dilutions.

The utility model discloses a fluorescence immunochromatographic test strip 100 has optimized the position of detection line 42 for improving its linear scope, makes reaction film 40 be close to the one end of combination pad 30 and the distance between the detection line 42 be 1.35cm ~ 1.85cm, is favorable to increasing the linear scope of test strip. The principle is that when the sample is specifically combined with the labeled antibody on the binding pad 30, an antigen-antibody fluorescent complex is formed, and within a certain range, when the detection line 42 is close to the binding pad 30, the reaction time of the sample and the labeled antibody is shortened, the number of the labeled antibodies which can be captured is reduced, the signal is weakened, and the increase of the linear range is facilitated; when the detection line 42 is far away from the end of the binding pad 30, the reaction time between the sample and the labeled antibody is increased, the amount of the labeled antibody that can be captured is increased, and the signal is strengthened, which is not favorable for increasing the linear range. Proved by experiments, the fluorescence immunochromatographic test strip 100 of the utility model can reliably measure the SAA concentration in a very wide concentration range (8 mg/L-1000 mg/L), thereby distinguishing horses with different inflammatory states, being used for monitoring the change of inflammatory activity and having very high applicability in equine species.

In a specific example, the fluorescence immunochromatographic test strip 100 further includes a blocking pad 60 disposed on the base plate 10, the sample pad 20, the blocking pad 60, the conjugate pad 30, the reaction membrane 40, and the absorbent pad 50 are sequentially overlapped, and the blocking pad 60 contains a blocking agent. By adding the blocking pad 60 and overlapping the binding pad 30, the non-specific protein in the sample can be blocked more thoroughly before contacting the labeled antibody, so that the foreign protein in the sample can be eliminated better, the non-specific binding of the foreign protein can be reduced, and the linear range can be further improved.

In a specific example, the length of the blocking pad 60 along the extending direction of the fluorescence immunochromatographic strip 100 is 0.8cm to 1.2cm, which helps to block non-specific proteins better.

In one specific example, the overlapping length of the blocking pad 60 and the bonding pad 30 is 1.5mm to 3 mm. In one particular example, the blocking pad 60 is a fiberglass pad or a polyester film pad. It will be appreciated that the specific type of blocking pad 60 is not limited thereto and may be selected as desired. Optionally, the blocking pad 60 and the conjugate pad 30 have a thickness of 0.2mm to 0.4 mm.

In a specific example, the reaction membrane 40 is further provided with a quality control line 41, the detection line 42 is closer to the absorbent pad 50 than the quality control line 41, and the quality control line 41 is coated with a quality control antibody.

In one specific example, the distance between the quality control line 41 and the detection line 42 is 3mm to 5 mm.

Optionally, the length of the binding pad 30 along the extending direction of the fluorescence immunochromatographic strip 100 is 1cm to 2 cm. Optionally, the length of the absorbent pad 50 along the extending direction of the fluorescence immunochromatographic test strip 100 is 2cm to 3 cm.

Alternatively, the base plate 10 is a PVC plate having an adhesive on the surface thereof, thereby facilitating more convenient attachment of the fixed sample pad 20, the blocking pad 60, the conjugate pad 30, the reaction membrane 40, the absorbent pad 50, and the like.

In a specific example, the labeled antibody is a mouse SAA monoclonal antibody labeled by a fluorescent microsphere, the diameter of the fluorescent microsphere is 300 nm-500 nm, the fluorescent microsphere is stable under a ground state, and the fluorescent microsphere emits fluorescence of 500 nm-600 nm under the action of an excitation light source of 300 nm-500 nm.

In one embodiment, the sample pad 20 contains anti-erythrocyte antibodies and lectins, so that erythrocytes can be intercepted, interference of erythrocytes in immunochromatography detection can be eliminated, and the linear range can be improved.

The utility model discloses a fluorescence immunochromatography detects card, including fluorescence immunochromatography test paper strip 100 and cutting ferrule (not shown) as shown in figure 1, fluorescence immunochromatography test paper strip 100 is located the cutting ferrule, and one of them surface of cutting ferrule has sample application window and display window, and sample pad 20 is relative with the sample application window, and detection line 42 is relative with the display window.

The utility model discloses a fluorescence immunoassay chromatography detects card accessible dry-type fluorescence immunoassay appearance quantitative determination fluorescence signal, its sensitivity is high, precision is good, the specificity is high, linear range is wider, detection speed is fast (5min can), easy operation and economical and practical can realize the accurate quantitative determination of horse SAA in serum, plasma, whole blood (EDTA-K2 anti-freezing pipe).

It will be appreciated that the control lines 41 are also opposite the display window. Optionally, the print window is circular and the display window is rectangular, but not limited thereto.

The utility model relates to a fluorescence immunochromatographic assay kit, including above-mentioned fluorescence immunochromatographic assay test paper strip 100 and sample diluent, the sample diluent contains 0.1 wt% -0.5 wt% casein sodium salt, 0.25M ~ 1.0M glycine, 0.1M ~ 0.3M sodium chloride, 0.5 wt% -1 wt% PVP-100, 0.5 wt% -1.5 wt% tween 80, 0.01M ~ 0.02M pH7.2's PB buffer solution and 0.05 wt% -0.1 wt% sodium azide.

The following are specific examples.

1. Preparation of detection kit

1.1 preparation of latex antibodies

1.1.1 labelling of SAA monoclonal antibodies

1) Cleaning fluorescent latex particles: adding 1mL of fluorescent latex particles into a 2mL centrifuge tube, centrifuging for 10min at 14000g, removing supernatant, and taking 1mL of solution A, wherein the solution A is 0.1M MES (2- (N-morpholine) ethanesulfonic acid) buffer solution and has the pH value of 6.0. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times.

2) Activation of the microspheres: adding 50-150 mu L of activator A and 50-150 mu L of activator B into the centrifuge tube in the step 1), uniformly mixing by vortex, and reacting for 20min on a rotary culture instrument.

The activator A is 0.05M MES buffer containing 20-80 mg/mL N-hydroxysuccinimide (NHS), and the pH is adjusted to 6.0-7.4. The activating agent B is 0.05M MES buffer solution containing 20-80 mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).

3) Activating and cleaning: centrifuging 14000g of activated latex particles for 10min, removing supernatant, taking precipitate, adding 1mL of cleaning solution which is 0.05M MES buffer solution, and adjusting the pH value to 6.0-7.4. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times.

4) Coupling antibody: and (3) after the ultrasonic treatment in the step 3), adding 20-60 mu g of horse SAA antibody, uniformly mixing by vortex, placing on a rotary incubator for reacting for 2 hours, after the reaction is finished, centrifuging for 10min at 14000g, centrifuging to remove supernatant, and taking out precipitate.

5) And (3) sealing: after the ultrasonic treatment in the step 4) is finished, adding 1mL of sealing liquid, wherein the sealing liquid is 0.05M TRIS buffer solution, 0.1-0.5M ethanolamine and 0.5-2 wt% casein, and adjusting the pH to 8.0. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times. Vortex and mix well, place on rotary incubator for 1 hour.

6) And (3) storage: after the reaction, 14000g of the solution was centrifuged for 10min, and the supernatant was removed by centrifugation to leave the precipitate. Adding 1mL of labeled latex particle preservative solution, carrying out ultrasonic resuspension for 2s, carrying out interval for 5s, and repeating for 5 times. The preservation solution contains 0.2-2 wt% of casein, 5-10 wt% of trehalose and 0.1M BIS-tris with pH7.4-pH8.0.

1.1.2 labeling of quality control C-line antibodies

1) Cleaning fluorescent latex particles: adding 1mL of latex particles into a 2mL centrifuge tube, centrifuging for 10min at 14000g, removing the supernatant, and taking 1mL of solution A, wherein the solution A is 0.1M MES (2- (N-morpholine) ethanesulfonic acid) buffer solution and has the pH value of 6.0. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times.

2) Activation of the microspheres: adding 50-150 mu L of activator A and 50-150 mu L of activator B into the centrifuge tube in the step 1), uniformly mixing by vortex, and reacting for 30min on a rotary culture instrument.

The activating agent A is N-hydroxysuccinimide (NHS) with the concentration of 20-80 mg/mL and 0.05M MES buffer (2- (N-morpholine) ethanesulfonic acid), and the pH is adjusted to 6.0-7.4. The activating agent B is 0.05M MES buffer (2- (N-morpholine) ethanesulfonic acid) containing 20-80 mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).

3) Activating and cleaning: centrifuging 14000g of activated latex particles for 10min, removing supernatant, taking precipitate, adding 1mL of cleaning solution which is 0.05M MES buffer solution (2- (N-morpholine) ethanesulfonic acid), and adjusting the pH value to 6.0-7.4. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times.

4) Coupling antibody: adding 5-20 mug of goat anti-rabbit antibody after the ultrasonic treatment in the step 3), uniformly mixing by vortex, placing on a rotary incubator for reacting for 2 hours, after the reaction is finished, centrifuging 14000g for 10min, centrifuging to remove supernatant, and taking out precipitate.

5) And (3) sealing: after the ultrasonic treatment in the step 4) is finished, adding 1mL of sealing liquid, wherein the sealing liquid is 0.05M TRIS buffer solution, 0.1-0.5M ethanolamine and 0.5-2 wt% casein, and adjusting the pH to 8.0. Ultrasonic resuspension, ultrasonic 2s, intermittent 5s, repeat 5 times. Vortex and mix well, place on rotary incubator for 1 hour.

6) And (3) storage: after the reaction, 14000g of the solution was centrifuged for 10min, and the supernatant was removed by centrifugation to leave the precipitate. Adding 1mL of labeled latex particle preservative solution, carrying out ultrasonic resuspension for 2s, carrying out interval for 5s, and repeating for 5 times. The preservation solution contains 0.2-2 wt% of casein, 5-10 wt% of trehalose and 0.1M BIS-tris with pH7.4-pH8.0.

1.2 preparation of blocking pad

Coating solution containing 3-5 wt% of trehalose, 0.05-0.2 wt% of sodium caseinate and 0.01M disodium hydrogen phosphate and having the pH value of 7.4 is used for mixing the following components: mouse sera were measured as 1: 40 and HBR-6 are diluted to 0.05-0.5 mg/mL and mixed together to be used as the working solution of the blocking pad. And 0.2-0.5 wt% of Evans blue is added into the blocking pad working solution, and the Evans blue is added into the blocking pad working solution, so that whether the spraying pad is uniform or not and whether a breakpoint occurs or not can be checked in production, the spraying leakage is prevented, and the precision of the test strip is prevented from being influenced. Cutting the glass fiber into 30 multiplied by 1.0cm, spraying the blocking pad working solution on the glass fiber at the concentration of 4-8 mu L/cm, and drying in a drying oven at 50 ℃ for 18-24 h to obtain the blocking pad.

1.3 preparation of conjugate pads

The SAA-labeled antibody and the quality control labeled antibody chicken IgY are respectively diluted to 0.5-5 mg/mL by using a fluorescent latex antibody coating solution with the pH value of 7.4 and containing 5-20 wt% of trehalose, 0.5-1 wt% of casein, 0.05-0.5 wt% of mannitol, 0.1-0.5 wt% of Tween, 0.02-0.5 wt% of PC-300 and 0.01M PB, and are mixed to be used as a working solution of a binding pad. And 0.2-0.5 wt% of Evans blue is added into the working solution of the combined pad, so that whether the sprayed pad is uniform or not and whether a breakpoint occurs or not can be checked in production, the spraying leakage is prevented, and the precision of the test strip is prevented from being influenced. Cutting the glass fiber into 30 multiplied by 1.6cm, spraying the bonding pad working solution on the glass fiber at the concentration of 4-8 mu L/cm, and drying the glass fiber in a drying oven at 50 ℃ for 18-24 h to obtain the bonding pad.

1.4 preparation of detection line and quality control line

1) And (3) diluting the other strain of SAA monoclonal antibody to 0.5-2 mg/mL by using a coating solution containing 3-5 wt% of trehalose and 0.01M sodium citrate and having the pH value of 7.4 to obtain the T-line working solution.

2) And (3) diluting the chicken IgY antibody to 0.5-1 mg/mL by using a coating solution containing 3-5 wt% of trehalose and 0.01M disodium hydrogen phosphate and having the pH value of 7.4 to obtain the C-line working solution.

3) Taking a PVC base plate, and sticking a nitrocellulose membrane.

4) And marking a quality control line C line and a detection line T line on the nitrocellulose membrane, sequentially, parallelly and adjacently spacing for 4mm, sequentially spacing and adjacently arranging the detection line at one end close to the combination pad and the quality control line, keeping the quality control line C line away from one end close to the water absorption pad of the combination pad, and marking the concentration of the lines to be 1-2 muL/cm.

5) And after the completion, drying the sheet in a drying oven at 50 ℃ for 24-72 h.

1.5 preparation of sample pad

1) Preparing a sample pad treatment solution: soaking the glass fiber in a PB buffer solution of 0.01M pH7.2 containing 0.2-0.5 wt% of casein and 1-5 wt% of trehalose for 0.5-2 h in a specification of 30 x 20cm, and drying the glass fiber in a drying oven at 50 ℃ for 18-24 h.

2) Cutting the glass fiber pretreated in the step 1) into 30 multiplied by 1.0cm after drying, preparing anti-RBC and agglutinin into 0.5-1 mg/mL working concentration serving as a sample pad working solution by using a coating solution containing 3-5 wt% of trehalose and 0.01M disodium hydrogen phosphate and having a pH value of 7.4, adding 0.2-0.5 wt% of Evans blue into the sample pad working solution to help production to check whether the sprayed pad is uniform or not, prevent break points and leakage and avoid influencing the precision of a test strip, spraying the sample pad working solution onto the glass fiber at a concentration of 4-8 muL/cm, and drying the glass fiber in a drying oven at 50 ℃ for 18-24 hours to obtain the sample pad.

1.6 preparation of absorbent pad

Cutting the absorbent pad into 30 × 2.4cm pieces.

1.7 test strip Assembly

The nitrocellulose membrane, the sample pad, the blocking pad, the combination pad and the water absorption pad are attached to a PVC plate, and the attached intermediate product is cut into test strips with certain width, so that the preparation is completed.

1.8 preparation of sample dilutions

Sample diluent: 0.1-0.5 wt% of casein sodium salt, 0.25-1.0M of glycine, 0.1-0.3M of sodium chloride, 0.5-1 wt% of PVP-100, 0.5-1.5 wt% of Tween 80, 0.01M of pH7.2 PB buffer solution and 0.05-0.1 wt% of sodium azide.

2. Assay kit assessment

2.1 comparison of the detection kit of this example with the conventional SAA detection kit

Preparing a horse SAA recombinant antigen reference substance, using horse negative serum as a substrate, respectively preparing the horse SAA recombinant antigen reference substance into 0mg/L, 7.8mg/L, 15.625mg/L, 31.25mg/L, 62.5mg/L, 125mg/L, 250mg/L, 500mg/L and 1000mg/L, comparing the conventional SAA detection kit with the detection kit of the embodiment, testing the same concentration strictly according to the using method of the control kit, and comparing the sensitivity and the linear range of the kit, wherein the results are shown in the following table and figure 2.

The results show that: the conventional SAA kit, which is a highly undesirable feature that can lead to an erroneously low concentration output, exhibits prozone effect (also referred to as hook effect) when the analyte is in excess and all antibodies in the assay are fully saturated, as compared to the kit of this example. The linear range of the kit is obviously superior to that of the conventional kit, and the linear range of the kit is 8 mg/L-1000 mg/L.

2.2 selection of the position of the detection line (T)

In this embodiment, the positions of the detection lines are optimized, and different positions are compared, where the positions are as shown in fig. 3: quality control line 1 bit-detection line 2 bit; 2-detection line 3 of quality control line; the quality control line is 1 bit-the detection line is 3 bits, and the detection method is the same as the above. As shown in FIG. 4, it was found that the quality control line was scribed at 1 position, the detection line was scribed at 2 positions, the quality control line was adjacent to the conjugate pad at a distance of 1.35cm, the detection line was adjacent to the conjugate pad at a distance of 1.75cm, the quality control line was adjacent to the detection line at a distance of 0.4cm, and the linearity range was 8-1000 mg/L.

2.3 optimization of sample dilutions

The conditions of not adding glycine and casein sodium salt, independently adding glycine, independently adding casein sodium salt and mixing and adding glycine and casein sodium salt in the sample diluent formula are compared, and the detection method is the same as that described above. Preparing a horse SAA recombinant antigen reference substance, using horse negative serum as a substrate, respectively preparing the horse SAA recombinant antigen reference substance into 0mg/L, 7.8mg/L, 15.625mg/L, 31.25mg/L, 62.5mg/L, 125mg/L, 500mg/L and 1000mg/L, respectively taking 10 microliters of each calibrator, adding 1000 microliters of diluents with different formulas to mix uniformly, taking 75 microliters of diluents by a pipetting gun, dropwise adding the diluents into a sample adding hole for measurement, wherein the concentration of the horse SAA recombinant antigen reference substance and the corresponding T/C value are shown in the table, and a linear graph is established by taking the average value of T/C as a vertical coordinate and the horse SAA recombinant antigen reference substance as a horizontal coordinate, and the results are shown in the table.

The results show that: the linear range of the mixed addition of the glycine and the casein sodium salt is obviously superior to the condition that the glycine and the casein sodium salt are not added, the glycine is independently added or the casein sodium salt is independently added, and compared with the-T/C mean value of a point 0, the addition of the glycine and the casein sodium salt is beneficial to reducing background signals, so that the linear range reaches 8-1000 mg/L.

2.4 fitting Linear equation

The kit prepared by the method is used for preparing horse SAA recombinant antigen reference substances, horse negative serum is used as a substrate and is respectively prepared into 0mg/L, 7.8mg/L, 15.625mg/L, 31.25mg/L, 62.5mg/L, 125mg/L, 500mg/L and 1000mg/L, 10 microliters of each calibrator is respectively taken and added into 1000 microliters of diluent for mixing uniformly, 75 microliters of the calibrator is taken and added into a sample adding hole for measurement by a pipette gun, and the concentration of the horse SAA recombinant antigen reference substances and corresponding detection limit signal values and quality control line signal values are shown in the following table. Wherein, the detection limit signal value and the quality control line signal value are respectively represented by T and C, the average value of T/C of each concentration is calculated, the average value of T/C is taken as a vertical coordinate, the horse SAA recombinant antigen reference is taken as a horizontal coordinate, an equation is established and a linear regression curve is fitted, and the linear regression curve is shown in figure 5.

The results of the linear fit equation show: in the detection range of 8-1000 mg/L of the kit, the SAA linear fitting correlation coefficient R is larger than 0.99.

2.5 precision

Randomly drawing the kit of the same batch number, taking two reference substances of 100mg/L and 800mg/L, and calculating the mean value of the measurement result of the sample and the variation Coefficient (CV) of the precision in the batch by 10 reagent cards of each concentration test, as shown in the following table.

The results show that: the kit has good batch precision, and the batch precision variation Coefficient (CV) of the reference substances for detecting the two concentrations is less than 15%.

2.6 accuracy

SAA reference samples with concentrations of 20mg/L, 100mg/L and 800mg/L were measured, and the mean and relative deviation of the sample measurements were calculated 3 times for each concentration, as shown in the following table.

The results show that: the kit has good accuracy, and the relative deviation Bias% of the reference products with 3 detected concentrations is within +/-15%.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only represent some embodiments of the present invention, and the description thereof is specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, without departing from the spirit of the present invention, several variations and modifications can be made, which are within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (10)

1. The fluorescent immunochromatographic test strip is characterized by comprising a bottom plate, and a sample pad, a combination pad, a reaction membrane and a water absorption pad which are arranged on the bottom plate, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially lapped, a detection line is arranged on the reaction membrane, the distance between one end, close to the combination pad, of the reaction membrane and the detection line is 1.35 cm-1.85 cm, a capture antibody is coated on the detection line, and the combination pad contains a labeled antibody.

2. The immunofluorescent chromatography test strip of claim 1, further comprising a blocking pad disposed on the bottom plate, wherein the sample pad, the blocking pad, the binding pad, the reaction membrane and the water absorption pad are sequentially overlapped, and the blocking pad contains a blocking agent.

3. The fluorescence immunochromatographic test strip according to claim 2, wherein the overlapping length of the blocking pad and the binding pad is 1.5mm to 3 mm.

4. The immunofluorometric chromatography test strip of claim 2, wherein the length of the blocking pad along the extension direction of the immunofluorometric chromatography test strip is 0.8cm to 1.2 cm.

5. The fluorescence immunochromatographic test strip according to claim 2, wherein the blocking pad is a glass fiber pad or a polyester film pad.

6. The fluorescence immunochromatographic test strip according to claim 1, wherein the reaction membrane is further provided with a quality control line, the detection line is closer to the water absorption pad than the quality control line, and the quality control line is coated with a quality control antibody.

7. The fluorescence immunochromatographic test strip according to claim 6, wherein the distance between the quality control line and the detection line is 3mm to 5 mm.

8. The immunofluorometric chromatography test strip of any one of claims 1 to 6, wherein the length of the binding pad in the extending direction of the immunofluorometric chromatography test strip is 1cm to 2cm, and the length of the water absorption pad in the extending direction of the immunofluorometric chromatography test strip is 2cm to 3 cm.

9. The fluorescence immunochromatographic test strip according to any one of claims 1 to 6, wherein the base plate is a PVC plate having an adhesive on the surface.

10. A fluorescence immunochromatographic test strip according to any one of claims 1 to 9, and a card sleeve, wherein the fluorescence immunochromatographic test strip is located in the card sleeve, one surface of the card sleeve has a sample application window and a display window, the sample pad is opposite to the sample application window, and the detection line is opposite to the display window.

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