TWI896469B - Rapid test kit device and method for manufacturing polyamidoamine-gold-antibody complex - Google Patents

Abstract

A rapid test kit device is provided, including: a sample pad, a gold conjugate pad, a reaction pad, and an absorbent pad, the gold conjugate pad including a plurality of polyamidoamine-gold-antibody complexes. The invention is further provided a method for manufacturing a polyamidoamine-gold-antibody complex, including: combining a plurality of polyamidoamines (Polyamidoamine, PAMAM) with a plurality of gold nanoparticles to form a polyamidoamine-gold complex; and binding a plurality of antibodies to the polyamidoamine-gold complex.

Description

Rapid screening reagent device and method for producing polyamidoamine-colloid gold-antibody complex

The present invention relates to a rapid reagent screening device and a method for producing a polyamidoamine-colloid gold-antibody complex.

A lateral flow immunoassay rapid screening reagent consists of a sample pad, a binding pad, a rapid screening membrane, and an absorbent pad. When the sample is added to the sample pad, the liquid molecules gradually migrate to the absorbent pad through the capillary action of the fibers in each layer. If the sample contains the analyte, the test line will show a line disappearance or a line development, depending on the principle of competitive or sandwich immunoassay.

However, in competitive lateral flow immunoassays, either too strong or too weak an affinity between the antibody and the antigen may affect the effectiveness of the reagent. In particular, when the affinity is lower than expected, the difference between a negative and a positive result may not be obvious, leading to erroneous interpretation of the test results.

Therefore, it is necessary to provide a novel and advanced rapid screening reagent device and a method for preparing a polyamidoamine-colloid gold-antibody complex to solve the above-mentioned problems.

The main purpose of the present invention is to provide a rapid screening reagent device and a method for manufacturing a polyamidoamine-colloid gold-antibody complex that can enhance the detection signal.

To achieve the above objectives, the present invention provides a rapid reagent screening device comprising: a sample pad, a colloidal gold pad, a reaction pad, and an absorption pad. The colloidal gold pad comprises a plurality of polyamidoamine-colloid-antibody complexes.

To achieve the above objectives, the present invention provides a method for preparing a polyamidoamine-colloidal gold-antibody complex, comprising the following steps: combining a plurality of polyamidoamines (PAMAM) with a plurality of colloids to form a polyamidoamine-colloidal gold complex; and combining a plurality of antibodies with the polyamidoamine-colloidal gold complex to form the polyamidoamine-colloidal gold-antibody complex.

1: Rapid screening test device

2: Sample to be tested

11: Sample pad

12: Glue pad

13: Reaction pad

14: Absorbent pad

20: Polyamidoamine-colloid-antibody complex

21: Polyamidoamine

22: Colloidal gold

23: Antibodies

24: Polyamidoamine-colloidal gold complex

30: Test line

31: Control Line

32: Antigen

L1: First Direction

Figure 1 is a schematic diagram of the structure of a preferred embodiment of the present invention.

Figure 2 is a partial enlarged view of Figure 1 on the adhesive pad.

Figure 3 is a partial enlarged view of the test line in Figure 1.

Figure 4 is a flow chart of a preferred embodiment of the present invention.

Figure 5 is a schematic diagram of the steps for preparing the polyamidoamine-colloid gold-antibody complex according to a preferred embodiment of the present invention.

Figure 6 shows the experimental results of the test sample of the present invention and a graph showing the relationship between the concentration of the test sample and the difference value.

Figure 7 shows the experimental results of the test sample using the prior art and a graph showing the relationship between the concentration of the test sample and the difference value.

The following examples illustrate possible implementations of the present invention and are not intended to limit the scope of protection sought by the present invention. The phrases "one" or "at least one" preceding any term herein do not limit the quantity; the term may also include "plurality" as needed. Such variations in quantity are also within the scope of protection sought, and are therefore warranted.

1 to 7 , which illustrate an embodiment of the present invention, the rapid screening reagent device 1 of the present invention includes a sample pad 11 , a colloidal gold pad 12 , a reaction pad 13 , and an absorption pad 14 . The colloidal gold pad 12 has a plurality of polyamidoamine-colloidal gold-antibody complexes 20 . Specifically, each polyamidoamine-colloidal gold-antibody complex 20 includes a plurality of polyamidoamines 21 (PAMAM), a plurality of colloidal golds 22, and a plurality of antibodies 23. Each colloidal gold 22 is bound to a plurality of polyamidoamines 21; each antibody 23 is bound to a plurality of colloidal golds 22; and each polyamidoamine 21 (PAMAM) is bound to a plurality of colloidal golds 22.

As shown in Figures 4 and 5 , the present invention further provides a method for preparing the aforementioned polyamidoamine-colloid-antibody complex, comprising the following steps: combining a plurality of polyamidoamines 21 (PAMAM) with a plurality of colloids 22 to form a polyamidoamine-colloid-AuNP complex 24 (PAMAM-AuNP); and combining a plurality of antibodies 23 with the polyamidoamine-colloid-AuNP complex 24 to form the polyamidoamine-colloid-AuNP complex 20. In this embodiment, the plurality of polyamidoamines 21 (PAMAM) are combined with the plurality of colloids 22 via a coupling agent, which is EDC/sulfo-NHS.

In this way, the multiple polyamidoamines 21 (PAMAM) bind the multiple colloids 22 together, increasing the amount of colloids 22 attached to each of the antibodies 23, thereby enhancing the detection signal. Furthermore, the analyte in the test sample 2 can interact with the multiple polyamidoamine-colloid-antibody complexes 20, resulting in a positive detection signal.

Specifically, the sample pad 11, the colloid pad 12, the reaction pad 13, and the absorption pad 14 are arranged sequentially along a first direction L1. In this embodiment, the reaction pad 13 is provided with a test line 30 (T line) and a control line 31 in sequence along the first direction L1. The test line 30 is provided with multiple antigens 32, and each polyamidoamine-colloid-antibody complex 20 can be bound to an antigen 32. In this embodiment, the reaction pad 13 is a nitrocellulose membrane, and the colloid pad 12 is made of fiberglass.

During use, the test sample 2 is dropped onto the sample pad 11. The sample 2 flows from the sample pad 11 toward the absorbent pad 14 through a capillary effect. When the sample 2 reaches the colloidal gold pad 12, the polyamidoamine-colloidal gold-antibody complex 20 flows to the test line 30 due to the capillary effect and binds to the multiple antigens 32 on the test line 30, causing the test line 30 to develop a color. The polyamidoamine-colloidal gold-antibody complex 20 amplifies the detection signal, making the color more pronounced and enhancing the positive-negative difference in the test results, thereby preventing false-negative or false-positive results.

In this embodiment, the polyamidoamine-colloid-antibody complexes 20 are spray-coated onto the metal pad 12. The polyamidoamine-colloid-antibody complexes 20 can bind to the test sample 2, allowing the polyamidoamine-colloid-antibody complexes 20 to be evenly distributed on the metal pad 12. The polyamidoamine-colloid-antibody complexes 20 are spray-coated onto the metal pad 12 to form a spray coating.

Preferably, each of the colloidal metals 22 is bonded to multiple opposite sides with at least one polyamidoamine (PAMAM) 21, allowing a single colloidal metal 22 to bond to multiple colloidal metals 22 in multiple directions. Specifically, the colloidal metal 22 is granular with a diameter ranging from approximately 10 nm to 100 nm, which facilitates bonding with at least one polyamidoamine (PAMAM) 21.

Figure 7 shows the test results of a prior art rapid screening reagent device 1. Figure 6 shows the test results of a rapid screening reagent device 1 according to the present invention. The experiment was conducted using a competitive immunoassay. To further illustrate, in this case (Figure 6), as the concentration of the test sample 2 (BE) gradually increased (from 0 ng/mL to 40 ng/mL), the color of the test line 30 on the rapid screening reagent device 1 gradually faded. In Figure 7 (prior art), when the test concentration is 0 ng/mL, the test line 30 is very pale and disappears when the test concentration is below the threshold, making it easy to mistakenly judge it as positive, resulting in a false positive. In contrast, Figure 6 shows the results of this case. The difference between positive and negative is obvious, making it less likely to misjudge. To further explain, the test threshold for the analyte is 20 ng/mL (the threshold is the legal test concentration; concentrations above the threshold are judged as positive, and concentrations below the threshold are judged as negative). Furthermore, comparing the curves of the relationship between the concentration and the difference value of the test sample 2 (BE) of the present invention in Figure 6 with the curves of the relationship between the concentration and the difference value of the test sample 2 (BE) of Figure 7 , the difference value of the concentration of the test sample 2 (BE) of similar concentration in the present invention is higher than the difference value of the prior art (Figure 7 ). Therefore, the detection signal intensity of the rapid screening reagent device 1 of the present invention is high.

1: Rapid screening test device

2: Sample to be tested

11: Sample pad

12: Glue pad

13: Reaction pad

14: Absorbent pad

30: Test line

31: Control Line

L1: First Direction

Claims (8)

A rapid screening reagent device includes a sample pad, a colloidal pad, a reaction pad, and an absorption pad. The colloidal pad comprises a plurality of polyamidoamine-colloid-antibody complexes. Each of the polyamidoamine-colloid-antibody complexes comprises a plurality of polyamidoamines (PAMAMs), a plurality of colloidal metals, and a plurality of antibodies. Each of the colloidal metals is bound to a plurality of polyamidoamines (PAMAMs). Each of the antibodies is bound to a plurality of colloidal metals. Each of the polyamidoamines (PAMAMs) is bound to a plurality of colloidal metals. The reaction pad is provided with a test line and a control line. The test line is provided with a plurality of antigens. The rapid screening reagent device as described in claim 1, wherein the plurality of polyamidoamine-colloid-antibody complexes are formed by spraying and covered on the colloidal pad. The rapid screening reagent device as described in claim 1, wherein the sample pad, the gold pad, the reaction pad and the absorption pad are arranged in sequence along a first direction. The rapid reagent screening device as described in claim 1, wherein at least one polyamidoamine (PAMAM) is bonded to multiple opposite sides of each of the colloids. The rapid screening reagent device as claimed in claim 3, wherein the reaction pad is provided with the test line and the control line in sequence toward the first direction A rapid screening reagent device as described in claim 1, wherein the plurality of polyamidoamine-colloid-antibody complexes are formed by spraying and coated on the colloidal pad; the sample pad, the colloidal pad, the reaction pad, and the absorption pad are arranged in sequence along a first direction; the reaction pad is provided with the test line and the control line in sequence toward the first direction; wherein each polyamidoamine-colloid-antibody complex can be bound to one of the antigens; and the colloidal pad is made of glass fiber material. A method for preparing a polyamidoamine-colloid-antibody complex comprises the following steps: combining a plurality of polyamidoamines (PAMAMs) with a plurality of colloids to form a polyamidoamine-colloid complex; and combining a plurality of antibodies with the polyamidoamine-colloid complex to form the polyamidoamine-colloid-antibody complex; wherein each of the polyamidoamine-colloid-antibody complexes comprises a plurality of polyamidoamines (PAMAMs), a plurality of colloids, and a plurality of antibodies; wherein each of the colloids is combined with a plurality of the polyamidoamines (PAMAMs). wherein each of the antibodies is conjugated to a plurality of the colloids; wherein the reaction pad is provided with a test line and a control line; wherein the test line is provided with a plurality of antigens; wherein each of the polyamidoamine (PAMAM) is conjugated to a plurality of the colloids. The method for preparing a polyamidoamine-colloid-antibody complex as described in claim 7, wherein the multiple polyamidoamines (PAMAMs) are bound to the multiple colloids via a coupling agent, and the coupling agent is EDC/sulfo-NHS.