CN118359697A - Beta amyloid 40 and beta amyloid 42 specific recognition polypeptide and application thereof - Google Patents

Abstract

The invention discloses a group of Abeta 40 and Abeta 42 specific recognition polypeptides, wherein the sequence of the Abeta 40 specific recognition polypeptide is shown as SEQ ID NO.1, and the sequence of the Abeta 42 specific recognition polypeptide is shown as SEQ ID NO. 2. The invention also discloses conjugates of the Abeta 40 and Abeta 42 specific recognition polypeptides and carrier proteins, application thereof, and an immunochromatography detection reagent or a kit for Alzheimer's disease prepared by the conjugates. Compared with the prior art, the magnetic particles coated or sealed by the polypeptide shown in SEQ ID NO.1 and SEQ ID NO.2 can effectively improve the sensitivity of the immunochromatography detection reagent under the condition of stable reagent components and process; furthermore, the polypeptides shown in SEQ ID No.1 and 2 do not or hardly cross-react with other types of amyloid beta, and can ensure both sensitivity and specificity.

Description

Beta amyloid 40 and beta amyloid 42 specific recognition polypeptide and application thereof

Technical Field

The invention belongs to the technical field of in-vitro diagnosis and detection, and in particular relates to a group of specific recognition polypeptides of beta-amyloid 40 and beta-amyloid 42 and application thereof.

Background

Alzheimer's disease is the most common neurodegenerative disease, the incidence of which has a progressive trend, called the first killer of the elderly's health. In recent years, the demand of early diagnosis reagent for Alzheimer's disease is higher and higher in China, and the existing main diagnosis schemes are cerebrospinal fluid detection, imaging diagnosis and the like, but the schemes have the characteristics of traumatism, high price and the like. In contrast, chemiluminescent and immunochromatographic techniques are the most popular diagnostic methods, and have the advantages of large detection throughput, low cost, no trauma, and the like. The bottleneck of applying chemiluminescence and immunochromatography to early blood diagnosis of Alzheimer's disease is mainly to improve the sensitivity and the precision of reagents.

Currently, the requirements for detecting the concentration of amyloid beta 40 (aβ40) and amyloid beta 42 (aβ42) in blood samples have approached the limit of the methodology for chemiluminescence sensitivity, and the concentration of other biomarkers of alzheimer's disease in blood, except aβ, is even lower, and the requirements for reagent antibodies, raw materials, processes and the like are increasingly higher. Therefore, the breakthrough in the aspects becomes the key point for improving the chemiluminescent sensitivity and providing an effective, cheap and rapid in-vitro diagnosis reagent for Alzheimer's disease. Existing or developing Alzheimer's disease blood immunodiagnosis reagents mainly depend on high-affinity antibodies as biological raw materials, and the concentration content of markers in blood is quantitatively analyzed through double-antibody sandwich methods. However, due to the large molecular weight of the antibody, the binding efficiency is limited in the solid-phase coating process, and the sensitivity of the reagent is affected to a certain extent due to the problem of antibody orientation in the coating process.

In summary, it is necessary to start from different routes and develop biological raw materials for immunodiagnosis including but not limited to antibodies, thereby further promoting the clinical diagnosis development of Alzheimer's disease and other neurodegenerative diseases.

Disclosure of Invention

The invention aims to improve the existing immunochromatography detection method for early clinical diagnosis of Alzheimer's disease so as to improve the detection sensitivity of the Alzheimer's disease blood markers Abeta 40 and Abeta 42.

In order to achieve the above object, according to a first aspect of the present invention, a set of aβ40 and aβ42 specific recognition polypeptides is provided, wherein the sequence of the aβ40 specific recognition polypeptide is shown in SEQ ID No.1, and the sequence of the aβ42 specific recognition polypeptide is shown in SEQ ID No. 2.

In a second aspect of the present invention, a group of conjugates of aβ40 and aβ42 specific recognition polypeptides and carrier proteins is provided, wherein the sequence of the aβ40 specific recognition polypeptide is shown in SEQ ID No.1, and the sequence of the aβ42 specific recognition polypeptide is shown in SEQ ID No. 2.

In a third aspect, the invention provides an application of the group of Abeta 40 and Abeta 42 specific recognition polypeptides in preparing an immunochromatography detection reagent or a kit for Alzheimer's disease, wherein the polypeptide shown in SEQ ID NO.1 is used for detecting Abeta 40, and the polypeptide shown in SEQ ID NO.2 is used for detecting Abeta 42.

According to the present invention, the use is to coat magnetic particle reagents with conjugates of the polypeptides shown in SEQ ID No.1 and 2 with carrier proteins, mix with the magnetic particle reagents coated with Abeta 40 and Abeta 42 antibodies, respectively, and then use the mixture in immunochromatographic assay reagents or kits.

According to a preferred embodiment of the present invention, the magnetic particle reagent coating the conjugate of the polypeptide shown in SEQ ID NO.1 and 2 and the carrier protein is mixed with the magnetic particle reagent coating the Abeta 40 or Abeta 42 antibody in a volume ratio of 1:4.

According to the invention, the magnetic particles coated with the conjugates of the polypeptides shown in SEQ ID No.1 and 2 and the carrier protein and the magnetic particles coated with the antibodies Abeta 40 and Abeta 42 are blocked by a conventional blocking buffer.

According to a preferred embodiment, the magnetic particles coated with the conjugate of the polypeptide shown in SEQ ID NO.1 and 2 with a carrier protein are blocked with a conventional blocking buffer; the blocking buffer solution for blocking the magnetic particles coated with the Abeta 40 antibody is a conjugate of the polypeptide of SEQ ID NO.1 and carrier protein which is added with 0.05 plus or minus 0.01 weight percent on the basis of the conventional blocking buffer solution; the blocking buffer solution for blocking the magnetic particles coated with the Abeta 42 antibody is a conjugate of the polypeptide of SEQ ID NO.2 and carrier protein which is added with 0.05 plus or minus 0.01 weight percent based on the conventional blocking buffer solution.

According to another preferred embodiment, the application is: adding a conjugate of the polypeptide shown in SEQ ID NO.1 and a carrier protein into a blocking buffer solution for blocking magnetic particles coated with the Abeta 40 antibody; and adding the conjugate of the polypeptide shown in SEQ ID NO.2 and the carrier protein into a blocking buffer solution for blocking the magnetic particles coated with the Abeta 42 antibody.

Further, the addition amount of the conjugate of the polypeptide shown in SEQ ID NO.1 or 2 and the carrier protein is 0.05+/-0.01 wt%.

In a fourth aspect of the invention, there is provided a magnetic particle blocking buffer, wherein 0.05+ -0.01% of a conjugate of a polypeptide shown as SEQ ID NO.1 or 2 and a carrier protein is added to the blocking buffer in percentage by weight.

In a fifth aspect of the present invention, there is provided an immunochromatographic assay reagent or kit for Alzheimer's disease, comprising a magnetic particle reagent coated with a conjugate of the polypeptides represented by SEQ ID No.1 and 2 and a carrier protein, wherein the polypeptide represented by SEQ ID No.1 is used for detection of Abeta 40 and the polypeptide represented by SEQ ID No.2 is used for detection of Abeta 42.

In a sixth aspect of the present invention, there is provided an immunochromatographic assay reagent or kit for Alzheimer's disease, comprising a magnetic particle reagent coated with an Abeta 40 or Abeta 42 antibody, which is blocked using the magnetic particle blocking buffer described above.

The invention has the following beneficial effects:

1. Compared with the prior art, the magnetic particles coated or blocked by the polypeptide shown in SEQ ID NO.1 and SEQ ID NO. 2 can effectively improve the sensitivity of the immunochromatography detection reagent under the condition of stable reagent components and process.

2. The polypeptides shown in SEQ ID No.1 and 2 of the invention have no or almost no cross reaction with other types of beta amyloid, and can ensure the sensitivity and the specificity at the same time.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The percentage concentrations referred to in the following examples are mass percentage concentrations unless otherwise specified.

In the following examples, polypeptide 1 shown in SEQ ID NO.1 (SEQ ID NO: SGYHHFAEDVGAIIGLMGGVV for detection of Abeta 40) and polypeptide 2 shown in SEQ ID NO.2 (SEQ ID NO: SGYHHFAEDVGAIMGVVIA for detection of Abeta 42) are both synthetic sequences, which are mainly aggregation core sequences when the Alzheimer's disease biomarkers beta amyloid 40 (Abeta 40) and 42 (Abeta 42) form a deposited state.

During synthesis, NHS active groups are added to the N end and dissolved in DMSO respectively, and the concentration is 10mg/mL. After solubilization, the polypeptide was diluted to 1mg/mL with 20mM HEPES buffer (pH 8.0), followed by coupling with Bovine Serum Albumin (BSA), casein, human gamma globulin (hIgG) and the like at a molar ratio of 20:1, the coupling buffer being 20mM HEPES (pH 8.0), and the reaction was carried out overnight at 4 ℃. After the coupling, unreacted polypeptides were separated using a desalting column, and the concentration of the polypeptide-BSA conjugate was determined using the BCA method.

For direct coating, 20 μg of polypeptide per mg of magnetic microparticles is added; for blocking, 0.05.+ -. 0.01% of polypeptide-BSA polymer was added to the blocking buffer.

When polypeptide reactivity is verified, a series of samples with concentration diluted in gradient and 10 random normal human serum samples are detected, and according to the corresponding detection content, all the groups of the steps are consistent except for different magnetic particle addition ratios or different magnetic particle blocking buffers.

The measured samples are all compared with the blank buffer solution to eliminate systematic errors, so that sensitivity analysis is convenient.

In the examples below, HEPES, bovine Serum Albumin (BSA), casein (Casein-Na), tween-type emulsifiers and biological preservatives are commercially available unless otherwise specified. The sealed preservation solution is prepared by a conventional preparation method of mixed solution. Full-automatic chemiluminescence immunoassay equipment Shine i1910 for immunofluorescence detection is a commercial product.

Example 1 preparation of beta-amyloid detection reagent

1.1 Preparation of R1 reagent

In this example, the R1 reagent comprises: magnetic particles coated with antibody specific for aβ40 (magnetic particle 1), magnetic particles coated with antibody specific for aβ42 (magnetic particle 2), magnetic particles coated with polypeptide 1 (magnetic particle 3), and magnetic particles coated with polypeptide 2 (magnetic particle 4).

The preparation method of the R1 reagent comprises the following steps:

the Abeta 40 antibody was dialyzed overnight with 0.05mol/L sodium bicarbonate under reaction conditions of 4 ℃. The liquid is changed at least three times in the dialysis process. After the completion of dialysis, the Abeta 40 antibody was taken out, the concentration was measured, the volume was fixed to 1mg/mL with 0.05M sodium bicarbonate buffer, and the mixture was kept at 4 ℃. The magnetic particles to be prepared are washed by using 0.10mol/L MES buffer solution at least three times, the magnetic particles and the antibodies are added according to the amount ratio of 10.00-40.00 mu g A beta 40 antibodies added to each 1.00mg magnetic particle, the magnetic particles and the antibodies with the amounts to be prepared are added, and the magnetic particles coated with the Abeta 40 antibodies (magnetic particles 1) are obtained after rotating and reacting for 3-5 hours at room temperature.

According to the same manner as described above, the Abeta 40 antibody was replaced with the Abeta 42 antibody, the polypeptide 1-BSA conjugate and the polypeptide 2-BSA conjugate, respectively, to obtain a magnetic particle coated with the Abeta 42 antibody (magnetic particle 2), a magnetic particle coated with the polypeptide 1-BSA conjugate (magnetic particle 3) and a magnetic particle coated with the polypeptide 2-BSA conjugate (magnetic particle 4).

Two kinds of sealing buffer solutions are adopted to seal the obtained magnetic particles 1-4, and the method comprises the following steps:

The formulation of the two blocking buffers was as follows:

The blocking buffer 1 adopts a conventional blocking buffer formula, and is prepared from deionized water: tris 50mM, naCl50mM, BSA 0.5%, proclin-300.03%, EDTA 1%, tween-20.1%, casein 0.5%.

Formulation of blocking buffer 2: on the basis of blocking buffer 1, 0.05.+ -. 0.01% of polypeptide 1-BSA conjugate or polypeptide 2-BSA conjugate was additionally added.

Two sealing schemes are as follows:

Scheme one: the magnetic particle 1 coated with Abeta 40, the magnetic particle 2 coated with Abeta 42 antibody, the magnetic particle 3 coated with polypeptide 1-BSA conjugate and the magnetic particle 4 coated with polypeptide 2-BSA conjugate are blocked by using the blocking buffer 1, and the blocking reaction is carried out for 14 to 18 hours at the temperature of 4 ℃.

Scheme II: the magnetic particles 1 coated with aβ40 and the magnetic particles 2 coated with aβ42 antibodies were blocked with the blocking buffer 2, and the reaction was performed at 4 ℃ for 14 to 18 hours.

After the closure is completed, the magnetic particles are separated from the liquid portion by a magnetic separation plate, and the magnetic particles are washed at least 2 times with MES buffer. Transferring to reagent buffer solution to constant volume to 10mg magnetic particles/mL, recording as concentrated solution of corresponding closed magnetic particles 1-4, and preserving at 4deg.C for use. When in use, the components are fully and evenly mixed.

The formulation of the reagent buffer (deionized water formulation): tris 0.10mol/L, BSA 1.00%, naCl 0.90%, casein-Na 0.50%, proclin-300.10%, triton X-405.01%, bovine serum gamma globulin (BGG) 1.00%, tween-20.50%, EDTA 0.20%, antifoaming agent 0.10%, gentamicin sulfate 0.01%, and Sucrose 15.00%.

1.2 Preparation of R2 reagent

In this example, the R2 reagent is an Abeta 40/42 antibody labeled with acridinium ester, and the preparation process is as follows:

The Abeta 40/42 antibody was dialyzed overnight against 0.05mol/L sodium bicarbonate at 4 ℃. The liquid is changed at least three times in the dialysis process. After dialysis, the antibodies were removed, measured for concentration, and added with an appropriate amount of 0.05M sodium bicarbonate buffer as needed to control the antibodies at the appropriate final concentration, and then stored at 4 ℃.

A reaction concentration of 0.1mg of acridine ester was added to 1mg of the antibody, and the Aβ40/42 antibody and acridine ester were added to a brown reaction flask under dark conditions. Shake reaction at room temperature for 1 hour.

An aqueous solution of 10.00% glycine was prepared, and 1mL of glycine solution was added to the reaction flask at a reaction concentration of 1mg of Abeta 40/42 antibody per 1mg of Abeta in a dark environment, followed by addition of a glycine solution of a corresponding volume. Shake reaction for 1-2 hours at room temperature.

After the reaction is finished, a 3.5KD dialysis bag which is of a proper length and is soaked in ethanol solution is taken, all the liquid in all the reaction bottles is added into the dialysis bag, 0.05mol/L sodium bicarbonate buffer solution is used for dialysis under the condition of 4 ℃ and light shielding, at least three liquid changes are carried out in the dialysis process, and the liquid change interval of each liquid change is not less than 4 hours.

After the dialysis is completed, the concentrated solution of Abeta 40/42 antibody of acridinium ester is measured by BCA method, and a proper amount of glycerol is added and the concentration is calibrated.

Since acridinium esters are sensitive to light, they need to be stored at-20℃in the absence of light. Taking out at least 30min before use, placing in dark place and recovering to room temperature, and avoiding generating gas/liquid bubbles during vibration.

Example 2 reagent Performance assessment

2.1 Magnetic particles, polypeptide-BSA coupled magnetic particles mixing ratio

The purpose of this example was to screen the optimal mixing ratio of magnetic particles to polypeptide-BSA coupled magnetic particles.

Experimental protocol: in this example, nonspecific immunoreactions of the conjugates of the polypeptides of SEQ ID No.1 and SEQ ID No.2 with BSA (polypeptide 1-BSA conjugate and polypeptide 2-BSA conjugate) were confirmed using the R1 reagent (Abeta 40-coated magnetic particle 1, abeta 42 antibody-coated magnetic particle 2, polypeptide 1-BSA conjugate-coated magnetic particle 3, and polypeptide 2-BSA conjugate-coated magnetic particle 4) prepared in example 1, respectively. The magnetic particles 1 and2 were diluted to a preset concentration using calf serum, respectively, and the magnetic particles 1 or 2 were mixed with the magnetic particles 3 or 4, respectively, according to the volume ratio shown in table 1, and formulated with the reagent buffer described in 1.2, ensuring the same final concentration of the magnetic particles.

Table 1: magnetic particles: polypeptide-BSA coupled magnetic particle mixing ratio

Group number	Magnetic particles 1: magnetic particles 3	Magnetic particles 2: magnetic particles 4
			Control	Magnetic particle 1 (non-magnetic particle 3)	Magnetic particle 2 (nonmagnetic particle 4)
#1	1:1	1:1
			#2	2:1	2:1
#3	4:1	4:1
			#4	8:1	8:1
#5	1:2	1:2
			#6	1:4	1:4
#7	1:8	1:8

The detection scheme is as follows:

# 1-7 and control group relative luminescence intensities (RLU) of serially diluted samples were measured, 3 determinations/group were performed for each concentration point, and the mean value (M) of the relative luminescence intensities (RLU) obtained by the three determinations, and the ratio (i.e., P/N) of the mean value (M) to the mean value (M) of the blank value relative luminescence intensities (RLU) were calculated. At the same concentration, the higher the relative luminescence intensity (RLU) and the larger the P/N, the better the reagent performance under this condition.

The detection is carried out by adopting a full-automatic chemiluminescence instrument, and the following general detection mode is used:

Step one, adding 50 mu L of sample into a reaction tube matched with an instrument;

step two, adding a 50 mu L R1 reagent into a reaction tube matched with the instrument;

step three, adding a 50 mu L R2 reagent into a reaction tube matched with the instrument;

step four, after mixing, incubating for 15 minutes at 37.00+/-1.00 ℃;

Fifthly, automatically performing magnetic separation by an instrument, and discarding supernatant;

Step six, automatically adding matched pre-excitation liquid and excitation liquid into the instrument according to a program, and uniformly mixing and incubating;

And step seven, detecting the luminous intensity.

The results of the measurements are shown in Table 2-1 and Table 2-2.

Table 2-1: aβ40 immunodetection best proportion screening data

Table 2-2: aβ42 immunodetection best proportion screening data

As can be seen from the results of tables 2-1 and 2-2, in the A.beta.40/42 immunodetection, when the R1 reagent is mixed according to the ratio of magnetic particles 1 to 3 to 4:1, the detection signal value and the sensitivity of the A.beta.polypeptide of each concentration are obviously improved compared with the control under the condition that the background signal value does not obviously change. When the ratio of the two is varied, the relative luminescence intensity (RLU) is measured in comparison with the control group, but the discrimination is still improved to some extent. Meanwhile, as shown in the result data, for Abeta 40/42 immunodetection, especially Abeta 42 immunodetection, after the magnetic particles 2 and 4 are adjusted according to the ratio of 4:1, the P/N measured by the lowest concentration point (1 pg/mL) is obviously improved, and the reagent can be used for stably distinguishing the samples at the concentration point.

2.2 Evaluation of the Effect of different blocking buffers on reagent Performance

The purpose of this example was to evaluate the effect of different blocking buffers (blocking buffer 1 and blocking buffer 2) on the reagent performance, keeping the remaining conditions consistent.

Experimental protocol: as described in 2.1. The difference is that only the effect of the different blocking buffers (blocking buffer 1 and blocking buffer 2) on the magnetic particles 1 and 2 was examined in this experiment, and the magnetic particles 3 and 4 were not involved.

The detection mode is the same as 2.1.

The results of the detection are shown in tables 3-1 and 3-2.

Table 3-1: influence of A beta 40 immunodetection blocking buffer change on reagent performance

Table 3-2: influence of A beta 42 immunodetection blocking buffer change on reagent performance

As can be seen from the results in tables 3-1 and 3-2, the detection signal value and the sensitivity of each concentration of Abeta polypeptide are improved by about 30% after blocking with the blocking buffer 2, compared with the magnetic particles blocked with the conventional blocking buffer 1. Meanwhile, compared with the sealing buffer solution 1, after the sealing buffer solution 2 is adopted for sealing, the distinguishing degree of the magnetic particle reagent at the minimum concentration point is obviously improved, and the sealing buffer solution 2 is adopted for sealing, so that the reagent performance is obviously facilitated.

2.3 Sample detection Performance assessment

The purpose of this example is to evaluate the improvement in performance of the reagent test samples over the control group under optimal conditions.

Experimental protocol: according to the experimental conclusion of 2.1 and 2.2, the magnetic particles 1 and 2 coated with Abeta 40 and Abeta 42 prepared by using the blocking buffer 2 are mixed with the magnetic particles 3 and 4 coated with the polypeptide 1-BSA coupling agent and the polypeptide 2-BSA coupling agent respectively in a ratio of 4:1, and are prepared by using the reagent buffer of 1.2, so that 10 random samples are measured. The ratio (S/N) between the relative luminescence intensity (RLU) measured by the sample and the relative luminescence intensity (RLU) measured by the control was calculated using calf serum as the control. The stronger the relative luminescence intensity (RLU) is measured for the same sample, and the higher the S/N, the better the reagent performance. The evaluation setting group and the detailed conditions are shown in Table 4.

Table 4: reagent performance evaluation group settings

The detection mode is the same as 2.1.

The results of the detection are shown in tables 5-1 and 5-2.

Table 5-1: aβ40 immunodetection sample assay data

Table 5-2: aβ42 immunodetection sample assay data

As can be seen from the results of tables 5-1 and 5-2, in the A.beta.40/42 immunodetection reagent, the addition of the blocking buffer and the addition of the polypeptide 1-BSA or the polypeptide 2-BSA conjugate to the coated or blocked magnetic particles 3 or 4, and the measurement of the relative luminescence intensity (RLU) of the random sample as close to or higher than that of the control group, with the relative luminescence intensity (RLU) of the blank control being close to or slightly lower than that of the control group, indicates that the addition of the polypeptide 1-BSA or the polypeptide 2-BSA conjugate to the coated magnetic particles and the replacement of the blocking buffer of the magnetic particles 1/2 coated with A.beta.40/42 can improve the signal value and the sensitivity of the reagent detection.

2.4 Evaluation of precision

The purpose of this example was to evaluate the difference in precision between the control and the control group under optimal conditions.

Experimental protocol: sample dilution buffer or calf serum was tested, and the same vial of buffer or calf serum was repeatedly tested 20 times for each reagent set, the mean (M) and Standard Deviation (SD) of the relative luminescence intensity (RLU) were calculated, and the coefficient of variation (CV%) was calculated.

When the conditions other than the blocking buffer were unchanged, the reagent measured the mean value (M) of the relative luminescence intensity (RLU) of the sample dilution buffer or calf serum and the smaller the coefficient of variation (CV%) was, the better the reagent performance was. The evaluation group settings are as shown in table 4 in 2.3.

The detection mode is the same as 2.1.

The results of the measurements are shown in tables 6-1 and 6-2.

Table 6-1: experimental data for evaluating repeatability of Abeta 40 immunodetection reagent

Table 6-2: experimental data for evaluating repeatability of Abeta 42 immunodetection reagent

From the results in tables 6-1 and 6-2, it is seen that the group to be evaluated exhibited the same reproducibility in the A.beta.40/42 immunoassay, while the reagent reproducibility exhibited by magnetic particles 1 or 2 blocked with blocking buffer 2 and magnetic particles 3 or 4 added with polypeptide 1-BSA or polypeptide 2-conjugate was optimal.

2.5 Sensitivity evaluation

The purpose of this example was to evaluate the sensitivity difference between the control and the control group under optimal conditions.

Experimental protocol: the recombinantly expressed project antigen was diluted with calf serum and the gradient diluted samples were assayed 2 times per spot. The ratio (i.e., P/N) between the average value (M) of the relative luminescence intensity (RLU) measured at each point and the average value (M) of the relative luminescence intensity (RLU) measured with the buffer solution was calculated.

The stronger the relative luminescence intensity (RLU) and the higher the P/N, the better the reagent performance is indicated when the conditions other than the blocking solution are unchanged at the same concentration point. The evaluation group settings are as shown in table 4 in 2.3.

The detection mode is the same as 2.1.

The results of the measurements are shown in tables 7-1 and 7-2.

Table 7-1: experimental data for evaluating sensitivity of Abeta 40 immunodetection reagent

Table 7-2: experimental data for evaluating sensitivity of Abeta 42 immunodetection reagent

As can be seen from the results of tables 7-1 and 7-2, the Abeta 40/42 immunodetection reagent can maintain a better signal for samples with different gradients, and the overall signal level is slightly lower than that of the control, but the overall discrimination is good. The #3 group of magnetic particles 1 or 2 blocked by the blocking buffer 2 and added with the magnetic particles 3 or 4 of the polypeptide 1-BSA or the polypeptide 2-conjugate has good sensitivity and distinguishing degree in each concentration section due to high overall signal level under the condition that the background is similar to the rest groups.

2.6 Evaluation of Cross-reactivity

The purpose of this example was to evaluate whether cross-reactivity was present between the A.beta.40/42 project-implemented group and the control group and the A.beta.42/40 protein under optimal conditions.

Experimental protocol: the recombinant expressed antigen was diluted to 10pg/mL with calf serum, and 0, 10, 30, 100, 300, 1000pg/mL of the antigen of the present invention was added thereto for detecting the Abeta 42/40 polypeptide 1/2, and the detection signal and the discrimination were affected by the measurement.

When the concentration of the 1/2 addition of the polypeptide is less than or equal to 100pg/mL, the absolute value of the deviation of the measured relative luminous intensity (RLU) from the point where the concentration of the 1/2 addition of the polypeptide is 0pg/mL (i.e. no polypeptide is added) is not more than 5%. The evaluation group settings are as shown in table 4 in 2.3.

The detection mode is the same as 2.1.

The results of the measurements are shown in tables 8-1 and 8-2.

Table 8-1: experimental data for evaluating cross-reactivity of Abeta 40 immunodetection reagent

Table 8-2: experimental data for evaluating cross-reactivity of Abeta 42 immunodetection reagent

As is clear from the results of tables 8-1 and 8-2, the respective concentrations of the Abeta 42/40 polypeptide 1/2 of the present invention were added to the diluted samples having the Abeta 40/42 concentration of 10pg/mL, and the degree of distinction between the standard and the background did not change significantly (i.e., the absolute value of the relative luminescence intensity (RLU) deviation was 5% or more) until the addition amount was 300 pg/mL. When the addition amount is 300pg/mL, 5-20% of the measurement result is affected; the addition amount of 1000pg/mL had an effect of 20 to 40% on the measurement results. The change in the differentiation at the remaining concentration is understood to be caused by the fluctuation of the instrument measurement and is not considered to have a significant influence on the reagent measurement result in this range of the added concentration.

The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of the present invention, but all equivalent changes or modifications according to the spirit of the present invention shall be included in the scope of the present invention.

Claims (12)

1. A group of Abeta 40 and Abeta 42 specific recognition polypeptides are characterized in that the sequence of the Abeta 40 specific recognition polypeptide is shown as SEQ ID NO.1, and the sequence of the Abeta 42 specific recognition polypeptide is shown as SEQ ID NO. 2.

2. A group of conjugates of Abeta 40 and Abeta 42 specific recognition polypeptides and carrier proteins are characterized in that the sequence of the Abeta 40 specific recognition polypeptide is shown as SEQ ID NO.1, and the sequence of the Abeta 42 specific recognition polypeptide is shown as SEQ ID NO. 2.

3. The use of a set of aβ40 and aβ42 specific recognition polypeptides as defined in claim 1 for the preparation of an immunochromatographic detection reagent or a kit for alzheimer's disease, wherein the polypeptide shown in SEQ ID No.1 is used for detection of aβ40 and the polypeptide shown in SEQ ID No.2 is used for detection of aβ42.

4. The use according to claim 3, wherein the use is to coat magnetic particle reagents with conjugates of the polypeptides shown in SEQ ID nos. 1 and 2 with carrier proteins, mix with the magnetic particle reagents coated with aβ40 and aβ42 antibodies, respectively, and then use the mixture in immunochromatographic assay reagents or kits.

5. The use according to claim 4, wherein the magnetic particle reagent coating the conjugate of the polypeptide shown in SEQ ID nos. 1 and 2 and the carrier protein is mixed with the magnetic particle reagent coating the aβ40 or aβ42 antibody in a ratio of 1:4 by volume.

6. The use according to claim 4, wherein the magnetic particles coated with the conjugate of the polypeptide shown in SEQ ID nos. 1 and 2 and the carrier protein and the magnetic particles coated with the antibodies aβ40 and aβ42 are both blocked with a conventional blocking buffer.

7. The use according to claim 4, characterized in that:

the magnetic particles coated with the conjugate of the polypeptide shown in SEQ ID NO.1 and SEQ ID NO. 2 and carrier protein are blocked by a conventional blocking buffer;

The blocking buffer solution for blocking the magnetic particles coated with the Abeta 40 antibody is a conjugate of the polypeptide of SEQ ID NO.1 and carrier protein which is added with 0.05 plus or minus 0.01 weight percent on the basis of the conventional blocking buffer solution;

The blocking buffer solution for blocking the magnetic particles coated with the Abeta 42 antibody is a conjugate of the polypeptide of SEQ ID NO.2 and carrier protein which is added with 0.05 plus or minus 0.01 weight percent based on the conventional blocking buffer solution.

8. The use according to claim 3, characterized in that the use is:

Adding a conjugate of the polypeptide shown in SEQ ID NO.1 and a carrier protein into a blocking buffer solution for blocking magnetic particles coated with the Abeta 40 antibody; and

The conjugate of the polypeptide shown in SEQ ID No.2 and carrier protein is added into a blocking buffer for blocking the magnetic particles coated with the Abeta 42 antibody.

9. The use according to claim 8, wherein the amount of the conjugate of the polypeptide shown in SEQ ID No.1 or 2 and the carrier protein is 0.05±0.01wt%.

10. A magnetic particle blocking buffer solution is characterized in that 0.05+/-0.01% of conjugate of polypeptide shown as SEQ ID NO.1 or 2 and carrier protein is added into the blocking buffer solution according to weight percentage.

11. An immunochromatographic detection reagent or kit for Alzheimer's disease, which is characterized by comprising a magnetic particle reagent coated with a conjugate of polypeptides shown in SEQ ID No.1 and 2 and a carrier protein, wherein the polypeptide shown in SEQ ID No.1 is used for detecting Abeta 40, and the polypeptide shown in SEQ ID No.2 is used for detecting Abeta 42.

12. An immunochromatographic assay reagent or kit for alzheimer's disease, characterized in that the reagent or kit comprises a magnetic particle reagent coated with an antibody to aβ40 or aβ42, which is blocked using the magnetic particle blocking buffer of claim 10.