CN219935868U

CN219935868U - Kit for detecting IgG subtype antibody

Info

Publication number: CN219935868U
Application number: CN202321085356.0U
Authority: CN
Inventors: 李志强; 李丽玮; 乐嘉宜
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2023-05-08
Filing date: 2023-05-08
Publication date: 2023-10-31
Anticipated expiration: 2033-05-08

Abstract

The utility model provides a kit for detecting an IgG subtype antibody, which comprises test strips for detecting the IgG1, igG2, igG3 and IgG4 subtype antibodies respectively; the test strip comprises a PVC bottom plate, a sample pad, a blood filtering film, a reagent pad, a nitrocellulose film coating a quality control line and a test line and a water absorbing pad; the upper surface of the middle part of the PVC bottom plate is fixedly provided with a nitrocellulose membrane; the sample pad, the blood filtering membrane and the reagent pad are sequentially overlapped and fixed on the PVC bottom plate in the horizontal direction, and the reagent pad is overlapped with one end of the nitrocellulose membrane; the water absorption pad is lapped with the other end of the nitrocellulose membrane and is fixed on the PVC bottom plate. The kit is added with the anti-interference pad, can be used for absorbing residual red blood cells in a diffused liquid sample and combining residual red blood cell fragments in the diffused liquid sample, ensures that fluorescent signals of NC films are not affected during detection, can accurately identify IgG subtype antibodies on sensitized red blood cells, and provides more reliable and effective experimental data for disease prognosis and clinical medication effect evaluation.

Description

Kit for detecting IgG subtype antibody

Technical Field

The utility model relates to the technical field of immunodetection, in particular to a kit for detecting an IgG subtype antibody.

Background

Neonatal hemolytic disease (hemolytic disease ofthe newborn, HDN) is one of the most common acute hemolytic diseases in pediatrics, and is characterized in that maternal and infant blood groups are not matched, fetus red blood cells are stimulated to the mother, immune blood group antibodies are mainly formed in pregnant mother, igG type antibodies are mainly used, the IgG antibodies can enter the fetus through placenta, HDN is caused by destroying the fetus red blood cells, abortion, premature birth, stillbirth, hemolytic jaundice and severe anemia can occur in severe cases, and nucleation jaundice can be developed, early death of infants can be caused or irreversible nervous system sequelae can be left in survivors, and great pain is brought to the infants and families of the infants. The most frequently found hemolytic diseases of newborn with ABO blood group incompatibility in human blood group system are Rh blood group system, and HDN caused by other blood group systems is less common.

The detection of IgG antibody subtype of high-titer antibody pregnant women by using a microcolumn gel method of blood group serology has been reported, and the result shows that IgG1 or IgG3 subtype positive has higher hemolytic disease occurrence probability and relatively heavy illness state, and corresponding treatment is usually carried out actively; the positive of IgG2 or IgG4 subtype has low probability of hemolysis and light symptoms, and corresponding treatment can not be carried out. Therefore, igG subtype detection plays an important role in the diagnosis and treatment of ABO neonatal hemolysis.

However, it was found clinically that some of the symptoms were not consistent with the reported descriptions in pregnant women with high titers of antibodies, but the hemolysis was not severe even though the titers of IgG1 and IgG3 subtype antibodies were high, or the titers of IgG1 and IgG3 subtype antibodies were low, but the hemolysis was severe. In order to explore the reality of this part, therefore, considering that IgG subtype antibodies in serum and on sensitized erythrocytes are detected separately, the detection result of which part of antibodies is more reference value for evaluation, namely whether the titer of the IgG subtype antibodies on sensitized erythrocytes is a main factor which truly influences the severity of hemolysis of newborns.

The recommended erythrocyte dispersion antibody detection in the recommended proposal (II) of the post-partum immunohematology test of the hemolytic disease of the newborn is a liquid reagent tube centrifugation method, and the operation steps are as follows: 1) taking 1 branch of test tube, adding 200 μl of dispersion liquid, 2) adding 50 μl of standard red blood cells with the same form as ABO of the infant, mixing, 3) performing water bath sensitization at 37 ℃ for 1h, 4) taking out, washing with saline for 3 times, and finally sucking water for 1 time, wherein each tube is added with 100 μl of anti-globulin reagent, 5) centrifuging for 15s at 1000g, and observing the result with naked eyes.

The method can only carry out qualitative and relative quantitative detection, has great influence on human factors and is unfavorable for result interpretation. Thus, there is a need for an accurate method for detecting IgG subtype antibodies in red blood cell dispersions.

Disclosure of Invention

In order to overcome the defects in the prior art and to avoid the problem that NC membrane blockage can not be completed due to red blood cells and red blood cell fragments in the diffusion liquid, the utility model provides a kit for detecting IgG subtype antibodies based on a fluorescence nanotechnology.

In order to achieve the above purpose, the utility model adopts the following technical scheme:

in a first aspect the utility model provides a kit for detecting antibodies of the IgG subtype comprising test strips for detecting antibodies of the IgG1, igG2, igG3 and IgG4 subtype, respectively;

the test strip comprises a PVC bottom plate, a sample pad, a blood filtering film, a reagent pad, a nitrocellulose film coating a quality control line and a test line, and a water absorbing pad; the upper surface of the middle part of the PVC (polyvinyl chloride) bottom plate is fixedly provided with a nitrocellulose membrane; the sample pad, the blood filtering membrane and the reagent pad are sequentially overlapped and fixed on the PVC bottom plate in the horizontal direction, and the reagent pad is overlapped with one end of the nitrocellulose membrane; the water absorption pad is overlapped with the other end of the nitrocellulose membrane and is fixed on the PVC bottom plate;

the test line is coated with an anti-IgG 1 subtype antibody, an anti-IgG 2 subtype antibody, an anti-IgG 3 subtype antibody or an anti-IgG 4 subtype antibody.

Further, the test strip further comprises an anti-Red Blood Cell (RBC) anti-interference pad, wherein the sample pad, the blood filtering membrane, the anti-red blood cell anti-interference pad and the reagent pad are sequentially overlapped and fixed on the PVC base plate in the horizontal direction, and the reagent pad is overlapped with one end of the nitrocellulose membrane.

Further, the overlapping length among the sample pad, the blood filtering membrane, the anti-erythrocyte anti-interference pad, the reagent pad, the nitrocellulose membrane and the water absorbing pad in the test strip is 1 mm-5 mm.

Further, the anti-erythrocyte anti-interference pad is a glass fiber film treated by spraying an anti-RBC antibody solution.

Further, according to the direction from the sample adding end to the water absorbing pad, the left end of the sample pad and the left end edge of the PVC bottom plate are on the same vertical line; the right end of the water absorption pad and the right end edge of the PVC bottom plate are on the same vertical line.

Further, the quality control line is coated with a fluorescent particle-labeled goat anti-rabbit IgG antibody.

Further, the sample pad is a glass cellulose film or a nonwoven fabric.

Further, the reagent pad is a glass cellulose membrane.

Further, the water absorbing pad is water absorbing filter paper.

Further, the kit further comprises a shell in which the test strip is arranged, a sample adding hole is formed in the top of the shell corresponding to the sample pad, and an observation window is formed in the position corresponding to the nitrocellulose membrane and can be used for observing a quality control line and a test line.

Further, the opening of the sample adding hole is circular, elliptical, rectangular, square, triangular or star-shaped.

Further, the opening of the observation window is circular, elliptical, oval, rectangular or square.

Compared with the prior art, the utility model has the following technical effects:

the kit provided by the utility model is provided with the anti-interference pad on the test paper strip, can be used for absorbing residual red blood cells in a scattered liquid sample and combining residual red blood cell fragments in the scattered liquid sample, ensures that fluorescent signals of NC films are not affected during detection, can accurately identify IgG subtype antibodies on sensitized red blood cells, can replace an ELISA method to detect and accurately identify IgG subtype free in serum (plasma), improves the original qualitative level to the accurate quantitative level, and provides more reliable and effective experimental data for continuously monitoring curve change of the concentration of the IgG subtype and evaluating disease prognosis and clinical medication effects.

Drawings

FIG. 1 is a schematic diagram of a test strip for detecting IgG subtype antibodies according to an embodiment of the present utility model;

FIG. 2 is a top view of a housing for detecting a positive sample according to an embodiment of the present utility model;

wherein, 1-PVC bottom plate, 2-sample pad, 3-hemofilter, 4-anti-erythrocyte interference pad, 5-reagent pad, 6-nitrocellulose membrane, 7-matter control line, 8-test line, 9-absorbent pad, 10-casing, 11-sample hole, 12-observation window, the arrow indicates the chromatography direction.

Detailed Description

The utility model provides a kit for detecting an IgG subtype antibody based on a fluorescence nanotechnology. The present utility model will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present utility model, but the following examples do not limit the scope of the present utility model.

Referring to fig. 1, the present utility model provides a kit for detecting IgG subtype antibodies based on fluorescent nanotechnology, which includes test strips for detecting IgG1, igG2, igG3 and IgG4 subtype antibodies, respectively. Taking a test strip for detecting an IgG1 subtype antibody as an example, the test strip comprises a PVC bottom plate 1, a sample pad 2, a blood filtering membrane 3, a reagent pad 5, a nitrocellulose membrane 6 coated with a quality control line 7 and a test line 8, and a water absorbing pad 9, wherein the left end of the sample pad 2 and the left end edge of the PVC bottom plate 1 are positioned on the same vertical line according to the direction from a sample adding end to the water absorbing pad (from left to right), and the right end of the water absorbing pad 9 and the right end edge of the PVC bottom plate 1 are positioned on the same vertical line; the upper surface of the middle part of the PVC (polyvinyl chloride) bottom plate 1 is fixedly provided with (preferably stuck with) a nitrocellulose membrane 6; the sample pad 2, the blood filtering membrane 3 and the reagent pad 5 are overlapped and fixed on the PVC bottom plate 1 in the horizontal direction sequence (namely the chromatographic direction), and the right end of the reagent pad 5 is overlapped with one end of the nitrocellulose membrane 6; the left end of the water absorbing pad 9 is overlapped with the other end of the nitrocellulose membrane 6 and is fixed on the PVC bottom plate 1;

wherein, the test line 8 is coated with an anti-IgG 1 subtype antibody, and the quality control line 7 is coated with a fluorescence particle labeled goat anti-rabbit IgG antibody. The test strips for detecting the IgG2, igG3 and IgG4 subtype antibodies have the same structure as the test strips for detecting the IgG1 subtype antibodies, except that the test strips are coated with the anti-IgG 2 subtype antibody, the anti-IgG 3 subtype antibody or the anti-IgG 4 subtype antibody respectively.

The hemofilter 3 is used for absorbing residual red blood cells in the scattered liquid sample. The blood filtering film is the existing product, and is cut into the size matched with the test strip. In a preferred embodiment of the utility model, the hemofilter uses BIOSET, BSPCY6316P.

In a preferred embodiment of the present utility model, the test strip further comprises an anti-erythrocyte anti-interference pad 4 disposed between the blood filtering membrane 3 and the reagent pad 5 for binding residual erythrocyte fragments in the bleeding liquid sample. Namely, the sample pad 2, the blood filtering membrane 3, the anti-erythrocyte anti-interference pad 4 and the reagent pad 5 are sequentially overlapped and fixed on the PVC base plate 1 in the horizontal direction, and the right end of the reagent pad 4 is overlapped with one end of the nitrocellulose membrane 6. The anti-erythrocyte anti-interference pad is a glass fiber film sprayed with anti-erythrocyte antibodies, and is cut to the matched size of the test strip.

In a preferred embodiment of the present utility model, the method for preparing the anti-erythrocyte anti-interference pad comprises the following steps:

a. preparing raw material of anti-RBC antibody (ORIGENE, product number: B9180RD 40-RD) from original concentration with antibody dilution (1 XPBS+0.1% trehalose) to working concentration of 0.5-1.5 mg/ml;

b. preparing 8951 glass fiber pretreatment liquid (100mM Tris+1%Tween-20+1%BSA+0.1%PVP) 50ml, uniformly wetting the whole 8951 glass fiber (osetron, product number: 7754313), and drying in a 45 ℃ oven for more than 12 hours after complete infiltration;

c. spraying antibody working solution on the pretreated 8951 glass fiber uniformly in a spraying amount of 6 mu l/cm by using a three-dimensional metal spraying film drawing instrument, and putting the glass fiber into a 45 ℃ oven for drying for 18-24 hours after the spraying is finished to prepare an anti-interference pad;

d. and cutting the dried anti-interference pad to a proper length and width.

In a preferred embodiment of the present utility model, the overlap length between the sample pad 2, the blood filter 3, the anti-erythrocyte anti-interference pad 4, the reagent pad 5, the nitrocellulose membrane 6 and the absorbent pad 9 in the test strip is 1mm to 5mm, that is, the overlap length between the sample pad 2 and the blood filter 3 is 1mm to 5mm, and accordingly, the overlap length between the blood filter 3 and the anti-erythrocyte anti-interference pad 4, the anti-erythrocyte anti-interference pad 4 and the reagent pad 5, the reagent pad 5 and the nitrocellulose membrane 6, and the nitrocellulose membrane 6 and the absorbent pad 9 is 1mm to 5mm, preferably 2mm or 3mm.

In a preferred embodiment of the present utility model, the sample pad 2 is a glass cellulose film or a nonwoven fabric. The sample pad needs to be pretreated by adopting the following sample pad pretreatment liquid: tris (Tris) 1.5143g, sodium caseinate 0.25g, tween-20 (Tween-20) 1.25mL, bovine Serum Albumin (BSA) 2.5g, proclin-3000.25mL, purified water to 250mL, and diluted hydrochloric acid lmol/L to adjust pH to 8.0+ -0.1.

In a preferred embodiment of the present utility model, the widths of the quality control wire 7 and the test wire 8 are the same, and the interval between the two is 3-8mm, preferably 5mm and 6mm.

In a preferred embodiment of the utility model, the reagent pad is a glass cellulose membrane coated with fluorescent particle-labelled anti-IgG 1 antibodies.

In a preferred embodiment of the present utility model, the absorbent pad is absorbent filter paper, and is disposed on the rightmost side of the test strip, for absorbing the redundant detection liquid.

In a preferred embodiment of the present utility model, referring to fig. 2, the kit further comprises a housing 10, the internal cavity of which is provided with the test strip. The top of the housing 10 is provided with a sample loading hole 11 corresponding to the sample pad 2 for receiving a sample to be tested (including but not limited to whole blood, red blood cell diffusing liquid, etc.), and an observation window 12 corresponding to the nitrocellulose membrane 6 for observing the quality control line 7 and the test line 8. In a preferred embodiment of the utility model, the opening of the well is circular, oval, rectangular, square, triangular or star-shaped, as shown in FIG. 2, preferably circular. In a preferred embodiment of the utility model, the opening of the viewing window is circular, oval, oblong, rectangular or square, as in fig. 2, preferably rectangular.

When the kit provided by the utility model is used for detection, only 4-6 drops of plasma, serum or red blood cell diffusion liquid are required to be added into a sample adding hole, the reaction is carried out for a period of time, and a test strip is inserted into a fluorescence immunoassay analyzer to read data.

According to the kit provided by the utility model, the anti-interference pad comprising the blood filtering membrane and the anti-erythrocyte anti-interference pad is arranged on the test strip, so that the fluorescent signal is not affected during detection, the sensitivity is high, the specificity is strong, the stability is good, the operation is convenient, multiple IgG antibody subtypes can be detected simultaneously, and the kit is suitable for large-scale popularization and use.

The above description of the specific embodiments of the present utility model has been given by way of example only, and the present utility model is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present utility model are intended to be within the scope of the present utility model. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present utility model without departing from the spirit and scope thereof.

Claims

1. The kit for detecting the IgG subtype antibody comprises test strips for detecting the IgG1, igG2, igG3 and IgG4 subtype antibodies, and is characterized by comprising a PVC bottom plate, a sample pad, a blood filtering film, a reagent pad, a nitrocellulose film coated with a quality control line and a test line, and a water absorbing pad; the upper surface of the middle part of the PVC bottom plate is fixedly provided with a nitrocellulose membrane; the sample pad, the blood filtering membrane and the reagent pad are sequentially overlapped and fixed on the PVC bottom plate in the horizontal direction, and the reagent pad is overlapped with one end of the nitrocellulose membrane; the water absorption pad is overlapped with the other end of the nitrocellulose membrane and is fixed on the PVC bottom plate;

the test line is coated with a fluorescent particle-labeled anti-IgG 1 subtype antibody, a fluorescent particle-labeled anti-IgG 2 subtype antibody, a fluorescent particle-labeled anti-IgG 3 subtype antibody, or a fluorescent particle-labeled anti-IgG 4 subtype antibody.

2. The kit of claim 1, wherein the test strip further comprises an anti-erythrocyte anti-interference pad, the sample pad, the blood filtering membrane, the anti-erythrocyte anti-interference pad and the reagent pad are sequentially overlapped and fixed on the PVC base plate in a horizontal direction, and the reagent pad is overlapped with one end of the nitrocellulose membrane.

3. The kit according to claim 2, wherein the overlap length between the sample pad, the blood filter, the anti-erythrocyte anti-interference pad, the reagent pad, the nitrocellulose membrane and the absorbent pad in the test strip is 1mm to 5mm.

4. The kit of claim 2, wherein the anti-erythrocyte anti-interference pad is a glass fiber membrane treated by spraying an anti-erythrocyte antibody solution.

5. The kit according to claim 1, wherein the left end of the sample pad is on the same vertical line as the left end edge of the PVC base plate and the right end of the absorbent pad is on the same vertical line as the right end edge of the PVC base plate in the direction from the sample addition end to the absorbent pad.

6. The kit of claim 1, wherein the quality control line is coated with a fluorescent particle labeled goat anti-rabbit IgG antibody.

7. The kit of claim 1, wherein the sample pad is a glass cellulose film or a nonwoven fabric; the reagent pad is a glass cellulose membrane; the water absorption pad is water absorption filter paper.

8. The kit according to claim 1, further comprising a housing in which the test strip is disposed, wherein a sample-applying hole is provided at a top of the housing corresponding to the sample pad, and an observation window is provided at a position corresponding to the nitrocellulose membrane, and the observation window is used for observing the quality control line and the test line.

9. The kit of claim 8, wherein the opening of the well is circular, oval, rectangular, square, triangular, or star-shaped.

10. The kit of claim 8, wherein the opening of the viewing window is circular, oval, oblong, rectangular or square.