CN118191313A

CN118191313A - Cat pancreatitis colloidal gold detection test strip, detection reagent pen and application thereof

Info

Publication number: CN118191313A
Application number: CN202410376162.9A
Authority: CN
Inventors: 徐晨骅; 张维宇; 董文坤
Original assignee: Hangzhou Anxiu Biotechnology Co ltd
Current assignee: Hangzhou Anxiu Biotechnology Co ltd
Priority date: 2024-03-29
Filing date: 2024-03-29
Publication date: 2024-06-14

Abstract

The invention provides a cat pancreatitis colloidal gold detection test strip, a detection reagent pen and application thereof, and relates to the technical field of in-vitro detection. According to the cat pancreatitis detection reagent pen, the outer edge of the detection pen core is vertically provided with the groove body which can be preloaded with the colloidal gold detection test strip, so that the colloidal gold detection test strip coated with the cat serum amyloid A monoclonal antibody, the cat amylase monoclonal antibody or the cat pancreatic lipase monoclonal antibody on the detection line is preloaded into the groove body. In the detection process, the multi-card detection can be realized on the basis of single sample mixing and single sample adding through the structural arrangement of the reagent pen, and the detection results of the cat serum amyloid A, the cat amylase and the cat pancreatic lipase can be obtained through one-time detection, so that the problem that repeated operation is often needed when the existing cat pancreatitis multi-joint detection sample adding is carried out or the problem that the multi-joint detection test strip is easy to have detection line misjudgment under the condition of common positive is effectively solved.

Description

Cat pancreatitis colloidal gold detection test strip, detection reagent pen and application thereof

Technical Field

The invention relates to the technical field of in-vitro detection, in particular to a cat pancreatitis colloidal gold detection test strip, a detection reagent pen and application thereof.

Background

The most common cause of cat pancreatitis is that a cat is fed with food with high fat content for a long time, or the cat is blocked by roundworms and the like to cause pancreatic ducts, and acute damage to pancreas, toxic and side effects of certain drugs and the like caused by infectious peritonitis, trauma or wound can possibly cause the occurrence of acute pancreatitis.

Cat serum amyloid A (f-SAA) is an acute phase protein. Especially in the early stages of the disease and tissue injury, serum amyloid a concentration may be raised 100-fold. Thus, serum amyloid a is the most effective marker of inflammation and produces the most rapid response in cats with a variety of inflammatory or infectious diseases. Serum amyloid A concentration in healthy cats is low, increases rapidly after inflammatory stimulus, reaches maximum concentration after 24-48 hours, and increases concentration by 100 times. Serum amyloid a is significantly increased in infections including keratoconjunctivitis, periodontitis, stomatitis, trauma, acute ophthalmia, acute pancreatitis, peritonitis, tympanic, ulceration, dermatitis; at the same time, serum amyloid a is also an effective index for assessing the presence of pancreatitis in cats. Since the cat serum amyloid A can be detected in early infection and is closely related to the severity and dynamic change of the disease, the cat serum amyloid A is an ideal marker in the clinical diagnosis and treatment process of infectious diseases.

Cat pancreatic amylase (fTL) is an index of pancreatic diseases, and its increase is seen in acute and chronic pancreatitis, pancreatic cancer, biliary tract diseases, gastric perforation, intestinal obstruction, parotitis, sialadenitis, and the like. It is found in liver diseases such as liver cancer and liver cirrhosis. Cat pancreatic amylase (fTL) was assayed along with LIPA (pancreatic lipase) to assess pancreatic disorders, with a large increase representing a potential for pancreatitis. Serum lipase is also elevated, which is indicative of kidney disease.

Cat serum pancreatic lipase (fPL) is an indicator of pancreatic disease. When cats suffer from pancreatitis, the level of cat pancreatic lipase in the blood increases dramatically. Pancreatic lipase is currently one of the best indicators for diagnosing cat pancreatitis specificity. Pancreatic lipase (fPL) and pancreatic amylase (fTL) are usually undetectable or low in concentration in blood, and are detected when pancreatic tissue is damaged, they enter the blood circulation.

In summary, it is difficult to detect and distinguish the symptoms of the digestive system and systemic reactions, which are not specific to cats suffering from pancreatitis. Among them anorexia (vomiting and diarrhea) and somnolence (very pain) are the most common symptoms. For cats, the risk of liver fat deposition (fatty liver) is present for more than 3 days of feeding. When inflammation spreads to the whole pancreas and other tissues or organs around, a series of complications are caused, so quantitative detection of cat pancreatitis markers can have great significance and great market demands on direct diagnosis, prognosis judgment and the like of diseases.

Since diagnosis of pancreatitis cannot be ascertained by a certain index, a joint detection of a plurality of items and reference are required in the diagnosis process. The application of the multi-joint detection technology in the auxiliary diagnosis of pancreatitis can greatly reduce the screening time in the detection process.

However, most of the existing multi-joint detection of cat pancreatitis is a double-window detection card or a multi-joint detection test strip, repeated operation is often required when the double-window detection card is used for sample feeding, and the pretreatment operation is complicated; the problem of misjudgment of the detection line is easy to occur under the condition of the co-positive of the multiple detection test strips, particularly when colloidal gold is used as a color development indication, the color development is consistent under the condition of the co-positive, and if a user misread the T line sequence, the diagnosis is wrong; meanwhile, most of the existing reagent kit detection test strips are arranged in a plate-type clamping shell, a sampling head is inserted into a lysate after sampling is completed, sampling is performed after incubation, the operation steps are complex, and risks of false negative are caused for common residents who are not in contact with medical equipment.

In view of this, the present invention has been made.

Disclosure of Invention

The first object of the present invention is to provide a colloidal gold test strip for detecting cat pancreatitis, which can detect cat serum amyloid a, cat amylase or cat pancreatic lipase by immunochromatography, respectively, and further can assist in diagnosis of cat pancreatitis by the combined detection of the above items.

The second object of the invention is to provide a cat pancreatitis detection reagent pen, which can realize multi-card detection on the basis of single sample mixing and single sample adding, can obtain detection results of 3 items of cat serum amyloid A, cat amylase and cat pancreatic lipase in one detection, and can judge the approximate degree of cat diseases according to the intensity of color development of a detection line by a tester, thereby effectively solving the problem that the false judgment of the detection line is easy to occur under the condition that repeated sample adding or multiple detection test strips are co-positive in the existing cat pancreatitis multiple detection.

The third aim of the invention is to provide an application of a colloidal gold detection test strip for detecting cat pancreatitis and a reagent pen for detecting cat pancreatitis in a cat pancreatitis detection product.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

According to one aspect of the invention, a cat pancreatitis colloidal gold test strip comprises a substrate, a sample pad, a label binding pad, an analytical membrane, and a water absorbing pad; the sample pad, the mark combining pad, the analysis film and the water absorbing pad are sequentially connected to the substrate according to the flowing direction of the sample;

the labeled binding pad is combined with a colloidal gold labeled detection antibody and an internal reference antibody;

The analysis film is sequentially provided with a detection line and a quality control line along the flowing direction of the sample;

The detection line is coated with one of a cat serum amyloid A monoclonal antibody, a cat amylase monoclonal antibody or a cat pancreatic lipase monoclonal antibody.

The application provides a cat pancreatitis colloidal gold detection test strip which comprises a substrate, a sample pad, a mark combination pad, an analysis film and a water absorption pad, wherein the sample pad is arranged on the substrate; wherein, the marked binding pad is combined with a colloidal gold marked detection antibody and an internal reference antibody; and the detection line and the quality control line are sequentially arranged on the analysis film along the flowing direction of the sample, and one of the cat serum amyloid A monoclonal antibody, the cat amylase monoclonal antibody or the cat pancreatic lipase monoclonal antibody is coated on the detection line. The colloidal gold detection test strip provided by the application can be used for respectively detecting cat serum amyloid A, cat amylase or cat pancreatic lipase by an immunochromatography method, and the combined detection results of the items can be used for carrying out auxiliary diagnosis on cat pancreatitis.

In a preferred embodiment of the present invention, the substrate of the catwalk colloidal gold test strip is a nitrocellulose membrane.

In a preferred embodiment of the present invention, the detection antibody labeled with colloidal gold on the labeled conjugate pad is a cat pancreatic lipase monoclonal antibody, and the concentration of the cat pancreatic lipase monoclonal antibody is 0.1-0.3 mg/mL.

In the above preferred embodiment, the colloidal Gold particles are selected from at least one of C-Gold1, C-Gold2 or C-Gold3, preferably C-Gold2;

Preferably, the particle size of the colloidal gold particles is in the range of 10 to 50nm.

In a preferred embodiment of the present invention, the label conjugate pad containing the colloidal gold label is prepared by spraying the diluted gold-labeled pad working fluid onto the gold-labeled pad.

Preferably, the components of the dilution liquid used for dilution include: 0.3g/L bovine serum albumin, 0.1g/L tris (hydroxymethyl) aminomethane, 0.2g/L sodium azide, 1g/L chloroauric acid and 3.3g/L trisodium citrate.

Preferably, the spraying is performed by adopting a metal spraying machine, and the metal is sprayed on the metal mark pad according to the thickness of 1.0-2.0 mu l/0.5-1 cm. More preferably 2.0. Mu.L/cm.

Preferably, before spraying, the gold-labeled pad is treated by a treatment liquid, and the treatment liquid comprises the following components: 7.11g/L disodium hydrogen phosphate, 1g/L bovine serum albumin, 4g/L polyvinyl alcohol and 3.3ml/L triton X-100.

In a preferred embodiment of the present invention, the assay membrane is provided with a detection line and a quality control line in sequence along the flow direction of the sample. The quality control line is coated with an internal reference quality control antibody which is specifically combined with the internal reference antibody;

Preferably, the internal reference quality control antibody coated on the quality control line is a goat anti-chicken IgY antibody, and the concentration of the goat anti-chicken IgY antibody is 0.1-0.3 mg/mL.

In the preferred embodiment, the concentration of the cat serum amyloid A monoclonal antibody coated on the detection line is 0.3-1.0 mg/mL;

Or, the concentration of the cat amylase monoclonal antibody coated on the detection line is 0.5-1.5 mg/mL;

or the concentration of the cat pancreatic lipase monoclonal antibody coated on the detection line is 0.5-1.5 mg/mL.

In a preferred embodiment of the present invention, the distance between the detection line and the quality control line is 0.3-0.5 cm, preferably 0.4cm.

In a preferred embodiment of the present invention, the sample pad is coated with a sample pad treatment liquid, the composition of the sample pad treatment liquid including: 1.1g/L sodium carbonate, 10g/L polyvinylpyrrolidone, 5g/L casein, 0.2g/L sodium azide, 0.1g/L tris (hydroxymethyl) aminomethane, murine anti-human RBC 0.3g/L and 15g/LTriton X; the pH value of the sample pad treatment solution is preferably 7.4+/-0.1.

As a preferable implementation mode, the sample pad treatment liquid has certain filtering and buffering effects on a detection sample, and can reduce the interference of the ionic strength and the pH value in the sample on a detection result; and can also reduce the non-specific binding of the sample. Therefore, after the sample pad treatment liquid is treated, the judgment of negative and positive in later detection is facilitated, and the accuracy and stability of an experimental result can be obviously enhanced.

According to one aspect of the invention, a reagent pen for detecting cat pancreatitis is provided, wherein the colloidal gold detection test paper strip for detecting cat pancreatitis is pre-loaded in the detection reagent pen.

In a preferred embodiment of the present invention, the detection reagent pen includes: the detection pen comprises a detection pen core, a reagent pen shell sleeved outside the detection pen core and a base;

the outer edge of the detection pen core is vertically provided with 3 groove bodies for pre-loading the catpancreatitis colloidal gold detection test paper strips, namely a first detection groove body, a second detection groove body and a third detection groove body, and vertical partitions are arranged among the groove bodies;

Wherein: the first detection groove body is preloaded with a first detection test strip, and a detection line of the first detection test strip is coated with a cat serum amyloid A monoclonal antibody;

The second detection groove body is pre-provided with a second detection test strip, and a detection line of the second detection test strip is coated with a cat amylase monoclonal antibody;

the third detection groove body is preloaded with a third detection test strip, and a detection line of the third detection test strip is coated with a cat pancreatic lipase monoclonal antibody;

the reagent pen shell is arranged in a transparent way, one end of the reagent pen shell is provided with a collecting part for collecting samples, and a groove body arranged on the outer edge of the detection pen core is communicated with the collecting part of the reagent pen shell;

The base is preloaded with a sample diluent.

According to the cat pancreatitis detection reagent pen provided by the application, through the groove body design of the colloidal gold detection test strip which is vertically arranged at the outer edge of the detection pen core and can be preloaded for cat pancreatitis detection, multi-card detection can be realized on the basis of single sample mixing and single sample adding, and detection results of cat serum amyloid A, cat amylase and cat pancreatic lipase items can be obtained through one-time detection, so that the problem that repeated operation is often required when the existing cat pancreatitis multi-joint detection sample adding is carried out or detection line misjudgment is easy to occur under the condition that the multi-joint detection test strip is co-positive is effectively solved.

In addition, the application leads the chromatography direction to be vertical from bottom to top through the improvement of the pen type, and the result can be observed in 5-8 min due to smaller colloidal gold compound particles in the chromatography process, and expensive instrument and equipment are not needed for detection, so that the detection of the pancreatitis of cats is simpler while the detection cost is effectively reduced, and the application is applicable to various detection scenes.

According to one aspect of the invention, the cat pancreatitis colloidal gold test strip or the cat pancreatitis test reagent pen is used for preparing cat pancreatitis test products.

The cat pancreatitis colloidal gold detection test strip or the cat pancreatitis detection reagent pen provided by the invention can be widely applied to the preparation process of cat pancreatitis detection products.

Compared with the prior art, the invention has the beneficial effects that:

The application provides a cat pancreatitis colloidal gold detection test strip which comprises a substrate, a sample pad, a mark combination pad, an analysis film and a water absorption pad, wherein the sample pad is arranged on the substrate; wherein, the marked binding pad is combined with a colloidal gold marked detection antibody and an internal reference antibody; and the detection line and the quality control line are sequentially arranged on the analysis film along the flowing direction of the sample, and one of the cat serum amyloid A monoclonal antibody, the cat amylase monoclonal antibody or the cat pancreatic lipase monoclonal antibody is coated on the detection line. The colloidal gold detection test strip provided by the application can be used for respectively detecting cat serum amyloid A, cat amylase or cat pancreatic lipase through an immunochromatography method, and further carrying out auxiliary diagnosis on cat pancreatitis through the combined detection result of the items.

According to the cat pancreatitis detection reagent pen provided by the application, through the groove body design of the cat pancreatitis colloidal gold detection test strip which is vertically arranged at the outer edge of the detection pen core and can be preloaded, multi-card detection can be realized on the basis of single sample mixing and single sample adding, and the detection results of cat serum amyloid A, cat amylase and cat pancreatic lipase items can be obtained through one-time detection, so that the problem that repeated operation is often required during the existing cat pancreatitis multi-joint detection sample adding or the problem that detection line misjudgment easily occurs under the condition that multiple detection test strips are co-positive is effectively solved.

The cat pancreatitis colloidal gold detection test strip and the cat pancreatitis detection reagent pen provided by the invention can obtain a detection result by detecting the whole blood/serum/plasma of suspected diseased cat, so that the cat pancreatitis can be diagnosed in an auxiliary way, and the cat pancreatitis colloidal gold detection test strip and the cat pancreatitis detection reagent pen can be widely applied to the preparation process of cat pancreatitis detection products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

Fig. 1 is a schematic diagram of the overall structure of a cat pancreatitis detection reagent pen provided in embodiment 4 of the invention;

Fig. 2 is a schematic diagram of the structure of a detection pen body of the cat pancreatitis detection reagent pen provided in embodiment 4 of the present invention.

Icon: 1-detecting a pen body; 2-a base; 11-detecting a pen core; 111-cartridge detection groove.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

A preparation method of a cat pancreatitis colloidal gold detection test strip specifically comprises the following steps:

(1) Preparing a colloidal gold label: colloidal gold was purchased from MAGSPHERE company, and the pH of the colloidal gold and goat anti-rabbit IgG polyclonal antibody solution was adjusted to 9.0 with 0.1Mol/L K ₂CO₃, respectively; the IgG solution is stirred electromagnetically, the colloidal gold solution is added, stirring is continued for 10min, and a certain amount of stabilizer is added to prevent the aggregation of the antibody protein and the colloidal gold from generating precipitation.

The added stabilizers were 5% fetal Bovine Serum (BSA) and 1% polyethylene glycol (molecular weight 20 KD). The added amount is as follows: 5% BSA gave a final concentration of 1%,1% polyethylene glycol was added to 1/10 of the total solution and the pH was adjusted to 8.2.

(2) Preparation of the label conjugate pad: diluting the rabbit IgG-colloidal gold marker obtained in the step (1) with a buffer solution, selecting a spraying amount of 2.0 mu L/cm by a gold spraying machine, and uniformly spraying on the treated marked binding pad.

The diluent comprises the following components: 0.3g/L bovine serum albumin, 0.1g/L tris (hydroxymethyl) aminomethane, 0.2g/L sodium azide, 1g/L chloroauric acid and 3.3g/L trisodium citrate.

The label bonding pad is firstly treated by polyester film treatment liquid, and the treatment liquid comprises the following components: 7.11g/L disodium hydrogen phosphate, 1g/L bovine serum albumin, 4g/L polyvinyl alcohol and 3.3ml/L triton X-100.

(3) Detection line antibody coating: diluting the raw materials of the quality control line and the detection line with PBS buffer solution;

The concentration of the cat serum amyloid A antibody coated on the detection line is 0.3mg/mL, and the cat serum amyloid A antibody is sprayed on an analysis film according to 1.0 mu l/cm;

the concentration of the goat anti-rabbit IgG antibody coated on the quality control line is 0.3mg/mL, and the goat anti-rabbit IgG antibody is sprayed on an analysis membrane according to 1.0 mu l/cm.

The analysis film is purchased from a PVC plate of CN95 of Sartolius, and is dried at 37 ℃ for 24 hours for standby to prepare a nitrocellulose film;

(4) Sample pad treatment preparation: glass fiber membranes were purchased (8964#) from aus corporation, sample pad treatment solutions were prepared in proportions, and the prepared solutions were then coated onto the fiber membranes, and then placed into a 37 ℃ oven for drying overnight for use.

The chemical components of the sample pad treatment fluid are as follows: 1.1g/L sodium carbonate, 10g/L polyvinylpyrrolidone, 5g/L casein, 0.2g/L sodium azide, 0.1g/L tris (hydroxymethyl) aminomethane, murine anti-human RBC 0.3g/L and 15g/LTriton X; the pH was 7.4.+ -. 0.1.

(5) And connecting the prepared sample pad, the label binding pad, the analysis membrane and the water absorption pad on the substrate in sequence according to the flowing direction of the sample to prepare the colloidal gold detection test strip for detecting the pancreatitis of cats.

Example 2

The cat pancreatitis colloidal gold test strip provided in this example is the same as that in example 1 except that the "cat serum amyloid a antibody" coated on the test line in step (3) is replaced with "cat pancreatic amylase antibody";

wherein, the concentration of the cat pancreatic amylase antibody coated on the detection line of the embodiment is 0.5mg/mL, and the cat pancreatic amylase antibody is sprayed on an analysis film according to 1.0 mu l/cm.

Example 3

The cat pancreatitis colloidal gold test strip provided in this example is the same as that in example 1 except that the "cat serum amyloid a antibody" coated on the test line in step (3) is replaced with "cat pancreatic lipase antibody";

Wherein, the concentration of the cat pancreatic lipase antibody coated on the detection line of the embodiment is 0.5mg/mL, and the cat pancreatic lipase antibody is sprayed on an analysis film according to 1.0 mu l/cm.

Example 4

Fig. 1 is a schematic diagram of the overall structure of a reagent pen for cat pancreatitis detection according to this embodiment.

Fig. 2 is a schematic structural diagram of a detection pen body 1 of a reagent pen for detecting cat pancreatitis according to this embodiment.

Referring to fig. 1 and 2, the cat pancreatitis detection reagent pen provided in this embodiment is composed of a detection pen body 1 and a base 2; wherein, the detection pen body 1 comprises a detection pen core 11 and a reagent pen shell sleeved outside the detection pen core 11.

During sampling, the sampling head at one end of the detection pen body 1 stretches into the serum/plasma collecting tube, and serum/plasma is sucked by capillary action. After the collection is finished, the detection pen sampling head is slowly and vertically inserted into the base 2 preloaded with sample diluent, the sample is diluted, the diluted sample is subjected to capillary force to be subjected to upward chromatography, and immunoreaction occurs after the diluted sample is contacted with a colloidal gold detection test strip preloaded in the pen core detection groove body 111 for detecting cat pancreatitis, the color development line intensity of the test strip is read, and the judgment result is interpreted according to the attached color chart or specification of the product, and the disease condition is defined.

Referring to fig. 2, in this embodiment, the outer edge of the detection cartridge 11 is vertically provided with 3 slots for preassembling colloidal gold detection test strips for detecting pancreatitis of cats, which are respectively a first detection slot, a second detection slot and a third detection slot, and vertical partitions are arranged between the slots;

wherein: the first detection groove body is preloaded with a detection test strip coated with the cat serum amyloid A antibody on the detection line provided in the embodiment 1;

The second detection groove body is preloaded with a detection test strip of which the detection line is coated with the cat pancreatic amylase antibody provided in the embodiment 2;

The third detection groove body is preloaded with the detection test paper strip coated with the catwalk lipase antibody on the detection line provided in the embodiment 3.

It should be noted that, the sample collection and result interpretation method of the cat pancreatitis detection reagent pen of this embodiment is as follows:

1. Sample collection and processing:

The test pen sampling head is extended into the serum/plasma collection tube to draw whole blood/serum/plasma by capillary action. After the collection is completed, the test pen sampling head is vertically inserted into the base 2 preloaded with the sample diluent.

Note that: the cat pancreatitis detection reagent pen can complete detection operation only by two steps of collecting and inserting the base 2 in the detection process, and effectively solves the problem that the detection steps are complex because repeated sample adding, detection and other operations are often required in the conventional cat pancreatitis multiplex detection.

2. Sample detection and result interpretation:

Low concentration antibody results: a band appears in the quality control region (C line), and the detection result is G4 and below in the detection region (T1 line). The results show that: the sample contains the corresponding detection result with low concentration.

Medium concentration antibody results: a band appears in the quality control area (C line), and in the detection area (T1 line), the detection result is G5-G6. The results show that: the sample contains a detection result corresponding to the medium concentration.

High concentration antibody results: a strip appears in the quality control area (C line), and the detection result is more than G6 in the detection area (T1 line). The results show that: the sample contains a high concentration corresponding detection result.

Invalidation: the absence of a mauve band in the quality control zone (line C) indicates incorrect handling or that the reagent has been spoiled, in which case a new reagent pen is used for re-detection.

Example 1 screening of different antibody concentration ratios of three-joint detection semi-quantitative detection reagent pens for cat pancreatitis

The test method comprises the following steps:

Referring to the specific embodiment, the test strip is prepared, in the antigen coating preparation process of the cat triplet antibody detection reagent, three different dilution concentration dilution detection line (T line) raw materials are selected and respectively diluted to 0.5mg/mL,1.0mg/mL and 1.5mg/mL, the rest preparation processes are kept consistent, and finally the test strip is assembled into a reagent pen for detection.

2. Test preparation of the detection reagent pen: taking a reagent pen for semi-quantitative detection of the three-joint detection of the cat pancreatitis, placing the reagent pen on a horizontal tabletop, standing and recovering to room temperature (15-25 ℃), taking a reagent pen for semi-quantitative detection of the three-joint detection of the cat pancreatitis, placing the table tabletop and standing to room temperature (15-25 ℃).

3. Preparing a sample to be tested: preparing a high-concentration cat serum amyloid a, cat pancreatic amylase and cat pancreatic lipase reference;

The cat pancreatic lipase reference was diluted to 0ng/mL, 3ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, respectively;

The cat serum amyloid A reference is diluted to 0ug/mL, 5ug/mL, 10ug/mL, 50ug/mL, 100ug/mL, 200ug/mL respectively;

cat pancreatic amylase reference samples were diluted to 0U/L, 100U/L, 200U/L, 500U/L, 1000U/L, 1500U/L, respectively.

4. And (3) detecting a sample and observing results: and (3) sucking 10 mu l of a sample by using a collecting tube on the pen point, vertically inserting the pen point into a cracking tube filled with sample diluent, standing on a table top for 10-15 minutes, and judging the result.

Table 1 cat pancreatitis triple assay semi-quantitative detection reagent pens different sample concentrations T-line chromogenic contrast:

Note that: in Table Fsaa, the cat serum amyloid A, FTL, cat pancreatic amylase and FPL are cat serum pancreatic lipase.

As can be seen by comparing the ratios of the concentrations of the T lines, when the ratio is carried out by using the concentration of the T line of 0.5mg/mL, the standard (G4) of the lowest detection limit of the concentration of the reference substance can be reached, so that the concentration sensitivity of the antibody with the concentration of the T line of 0.5mg/mL and the concentration of the T line of 0.3mg/mL can be obtained, and the gradient color development intensity is stronger. The concentration antibody with the T line concentration of 1.0mg/mL is used, false positive of a detection result is easy to cause, and the concentration antibody with the T line concentration of 0.5mg/mL is selected to achieve the best effect with less concentration and the highest titer in order to save the use cost.

Example 2 colloidal gold screening of semi-quantitative detection reagent pens for triple detection of cat pancreatitis

1. The test method comprises the following steps:

Referring to the method for preparing the test strip in the specific embodiment, three different types of C-emulsion, namely C-Gold1, C-Gold2 and C-Gold3, are selected in the process of preparing the emulsion microsphere label. After the colloidal gold is marked, the rest preparation processes are kept consistent, and finally the kit is assembled into a reagent pen for detection, and the contrast verification shows that the color development intensity of three different colloidal gold is different, and the sensitivity of the reagent is also different.

2. Preparing a sample to be tested: preparing a high-concentration cat serum amyloid a, cat pancreatic amylase and cat pancreatic lipase reference;

Table 2 cat pancreatitis triple assay semi-quantitative detection reagent pen different colloidal gold C/T line chromogenic contrast:

By comparing the types of latex particles used in the formula on the test strip, the color development intensity of the C-Gold2 colloidal Gold is stronger than that of C-Gold1 and C-Gold3, and the performance is optimal.

Example 3 sample pad treatment pH screening of cat pancreatitis Tri-joint detection semi-quantitative detection reagent pen

1. The test method comprises the following steps:

With reference to the preparation method of the test strip provided in the above specific embodiment, the preparation process of the sample pad is optimized. Selecting 8964# glass fiber membrane, preparing solution according to proportion, wherein the main chemical components of the solution are (0.1 mol of buffer salt, 0.8% polyvinylpyrrolidone (PVP), 0.5% casein and 1% surfactant), respectively preparing three parts of solution, adjusting pH to be pH=6.0, pH=7.4 and pH=9.0 respectively, coating the prepared solution on the fiber membrane, then placing the fiber membrane in a 37 ℃ oven for drying overnight, and keeping the rest preparation processes consistent.

2. Preparing a sample to be tested: preparing high-concentration cat serum amyloid A, cat pancreatic amylase and cat pancreatic lipase reference, and respectively diluting the cat pancreatic lipase reference to 0ng/mL, 3ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 200ng/mL; the cat serum amyloid A reference is diluted to 0ug/mL, 5ug/mL, 10ug/mL, 50ug/mL, 100ug/mL, 200ug/mL respectively; cat pancreatic amylase reference samples were diluted to 0U/L, 100U/L, 200U/L, 500U/L, 1000U/L, 1500U/L, respectively.

3. And (3) detecting a sample and observing results: and (3) sucking 10 mu L of a sample by using a collecting tube on the pen point, vertically inserting the pen point into a cracking tube filled with sample diluent, standing on a table top for 10-15 minutes, and judging the result.

Table 3 cat pancreatitis triple assay semi-quantitative detection reagent pen different pH sample pad contrast:

The results show that: the three pH sample pads were compared to simulate the corresponding buffer reaction environment. The sample pad after the slightly acidic treatment has the advantages of reduced contrast test sensitivity effect, low color development gradient and poor color development result, meanwhile, the sample pad after the slightly alkaline treatment has false positive condition in the color development process, and meanwhile, the color development result of the test strip is red, the overall movement speed is low, and the color development result is greatly influenced. The comparative analysis chooses to use a sample pad with ph=7.4.

Example 4 run plate speed comparison experiment of cat pancreatitis test reagent pen and existing test plate

The detection method comprises the following steps:

1. preparing a reagent: taking a cat pancreatitis triple detection semi-quantitative detection reagent pen, placing the reagent pen on a horizontal tabletop, standing and recovering to room temperature (15-25 ℃), taking a cat triple antibody detection reagent board, placing the table tabletop and standing to room temperature (15-25 ℃).

2. Preparing a sample to be tested: preparing a high-concentration cat serum amyloid a, cat pancreatic amylase, cat pancreatic lipase reference, and cat pancreatic lipase high-concentration reference; cat serum amyloid a high concentration reference; high concentration reference for cat amylopsin.

3. And (3) detecting a sample and observing results: the travel rate = L/t (L is the distance from line C to the bottom end of the sample pad, t is the time at which line C appears), the test pen sampling head is slowly inserted vertically into the sample diluent, the timing is stopped from the time of adding the sample by a stopwatch until the liquid reaches line C, the time taken is recorded as (t), and the length of line C to the bottom end of the sample pad is measured by a vernier caliper and recorded as (L).

The result shows that the speed of the running board of the sample detection pen is detected at 15.4mm/min, the speed of the running board of the sample detection pen is detected at 16.1mm/min, and the speeds of the running boards of the two products are basically consistent without obvious difference through the comparison of the speeds of the running boards of the cat pancreatitis triple detection semi-quantitative detection reagent pen and the detection board. Example 5 sensitivity contrast experiment of semi-quantitative detection reagent pen and detection plate for three-joint detection of cat pancreatitis

The detection method comprises the following steps:

3. And (3) detecting a sample and observing results: and (3) detecting a sample and observing results: and (3) sucking 10 mu l of a sample by using a collecting tube on the pen point, vertically inserting the pen point into a cracking tube filled with sample diluent, standing on a table top for 10-15 minutes, and judging the result.

TABLE 4 test results of sensitivity contrast of semi-quantitative detection reagent pen and reagent plate for triple detection of cat pancreatitis

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The result proves that the sensitivity of the cat pancreatitis triple detection semi-quantitative detection reagent pen is close to that of the detection plate and meets the detection requirement through comparing and verifying the detection result of the cat pancreatitis triple detection semi-quantitative detection reagent pen and the detection result of the detection plate.

Example 6 repeatability and stability experiment of cat pancreatitis triple detection semi-quantitative detection reagent pen

1. The test method comprises the following steps:

Preparing three batches of cat pancreatitis triple detection semi-quantitative detection reagent pens which are respectively marked as A1, B1 and C1, preparing high-concentration cat serum amyloid A, cat pancreatic amylase and cat pancreatic lipase references, and respectively diluting the cat pancreatic lipase references to 0ng/mL, 3ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 200 ng/mL; the cat serum amyloid A reference is diluted to 0ug/mL, 5ug/mL, 10ug/mL, 50ug/mL, 100ug/mL, 200ug/mL respectively; cat pancreatic amylase reference samples were diluted to 0U/L, 100U/L, 200U/L, 500U/L, 1000U/L, 1500U/L, respectively. Each concentration is repeated 5 times, and the mixture is respectively packaged in an aluminum foil bag with a drying agent, placed in an oven at 55 ℃ and taken out of 5 detection pens in 0 day, 3 days, 7 days and 14 days and 30 days respectively, so that on one hand, the accelerated stability is studied, and on the other hand, the indexes such as the repeatability, the sensitivity and the like of the reagent pens are detected.

2. Experimental results:

table 5 results of stability of cat pancreatitis triple assay semi-quantitative test reagent pen:

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The results prove that the cat pancreatitis triple detection semi-quantitative detection reagent pen has little batch-to-batch difference, the repeatability is 100 percent, and meanwhile, no false positive and false negative exist. The three samples developed can be kept stable at 55 ℃ for at least 30 days, and the reagent can be kept stable for 2 years at normal room temperature according to an Arrhenius formula (Arrhenius equation), and meanwhile, the sensitivity repeatability is stable and does not change greatly.

Example 7 clinical trial with semi-quantitative detection reagent pen for triple examination of cat pancreatitis

Five pet hospitals are randomly selected in Zhejiang area, 214 clinical cat blood samples are collected, the clinical samples are respectively detected by using the cat pancreatitis triple detection semi-quantitative detection reagent pen, a plastic cap is pulled out, a pen point is used for sucking 10 microliters of the samples, then the pen point is vertically inserted into a cracking tube filled with sample diluent, and a table top is placed for waiting for 10-15 minutes to judge results. The results are shown below.

The statistics of the above 214 clinical blood test results were performed based on the five pet hospitals clinical test results as a reference, as shown in table 6:

table 6 clinical test and test pen test results:

As can be seen from the corresponding test examples, the sensitivity of FSAA is 100%, the specificity is 90.9%, and the coincidence rate is 97.7%; the sensitivity of the FTL is 98.8%, the specificity is 90.6%, and the coincidence rate is 96.9%; the FPL sensitivity was 98.8%, the specificity was 86.3% and the compliance was 93.9%. Therefore, the cat pancreatitis detection reagent pen prepared by the invention accords with the existing pet hospital test result, the sensitivity and the specificity of the product are better, meanwhile, the detection reagent pen can realize multi-card detection on the basis of single sample mixing and single sample adding, and the detection results of cat serum amyloid A, cat amylase and cat pancreatic lipase items can be obtained by one-time detection, so that the problem that repeated operation is often required when the existing cat pancreatitis multi-joint detection sample adding is carried out or the detection line misjudgment is easy to occur under the condition that the multi-joint detection test paper strip is co-positive is effectively solved.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The cat pancreatitis colloidal gold detection test paper strip is characterized by comprising a substrate, a sample pad, a mark binding pad, an analysis film and a water absorption pad; the sample pad, the mark combining pad, the analysis film and the water absorbing pad are sequentially connected to the substrate according to the flowing direction of the sample;

2. The catwalk colloidal gold test strip according to claim 1, wherein the concentration of the catwalk serum amyloid a monoclonal antibody coated on the test line is 0.3-1.0 mg/mL;

3. The catwalk colloidal gold detection test strip of claim 1, wherein the quality control line is coated with an internal reference quality control antibody which specifically binds to the internal reference antibody;

4. The catwalk colloidal gold test strip according to claim 1, wherein the distance between the detection line and the quality control line is 0.3-0.5 cm, preferably 0.4cm.

5. The catwalk inflammation colloidal gold detection test strip according to claim 1, wherein the detection antibody marked by the colloidal gold on the marked binding pad is catwalk lipase monoclonal antibody, and the concentration of the catwalk lipase monoclonal antibody is 0.1-0.3 mg/mL.

6. The catwalk colloidal Gold test strip of claim 5, wherein the colloidal Gold particles are selected from at least one of C-Gold1, C-Gold2 or C-Gold3, preferably C-Gold2;

7. The catwalk colloidal gold test strip of claim 1, wherein the sample pad is coated with a sample pad treatment fluid;

The sample pad treatment solution mainly comprises sodium carbonate, polyvinylpyrrolidone, T casein, sodium azide, tris (hydroxymethyl) aminomethane and Triton X; the pH of the sample pad treatment solution was 7.4.+ -. 0.1.

8. A cat pancreatitis detection reagent pen, characterized in that the cat pancreatitis colloidal gold detection test strip according to any one of claims 1 to 7 is preloaded in the detection reagent pen.

9. The cat pancreatitis test agent pen of claim 8, wherein the test agent pen comprises: the detection pen comprises a detection pen core, a reagent pen shell sleeved outside the detection pen core and a base;

The outer edge of the detection pen core is vertically provided with 3 groove bodies for preassembling the cat pancreatitis colloidal gold detection test paper strip according to any one of claims 1 to 7, namely a first detection groove body, a second detection groove body and a third detection groove body, and vertical partitions are arranged between the groove bodies;

The base is preloaded with a sample diluent.

10. Use of the catwalk inflammation colloidal gold test strip according to any one of claims 1 to 7 or the catwalk inflammation test reagent pen according to claim 8 or 9 in catwalk inflammation test products.