CN111879923A

CN111879923A - Kit capable of eliminating HAMA effect

Info

Publication number: CN111879923A
Application number: CN202010787574.3A
Authority: CN
Inventors: 宁紫妍; 朱峰; 孙鸿华
Original assignee: Shenzhen Cologne Biological New Materials Co ltd
Current assignee: Shenzhen Cologne Biological New Materials Co ltd
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-11-03

Abstract

The invention provides a kit capable of eliminating HAMA effect, the technical scheme of the invention is that a humanized antibody for resisting antigen and a detection antibody with a biological catalytic enzyme labeled anti-humanized antibody are used, the detection antibody is connected with the humanized antibody in a universal way, the humanized antibody can eliminate false positive and false negative brought by HAMA effect in the antigen detection process, the universal detection antibody can reduce the labeling of the humanized antibody, so that the antigen detection kit is simpler in preparation method for different antigens, does not need to label each humanized antibody, and can be used for all antigen detections by only using the universal detection antibody for labeling.

Description

Kit capable of eliminating HAMA effect

Technical Field

The invention relates to the field of immunodetection, in particular to a kit capable of eliminating HAMA effect.

Background

The double antibody sandwich method is a common method for detecting antigen, and the method is to coat a capture antibody on a solid phase carrier, capture the antigen to be detected in a sample, combine a labeled antibody, and combine the capture antibody and the labeled antibody with different antigenic determinants to form a sandwich structure of capture antibody-antigen-labeled antibody.

Heterophily antibodies belong to the class of immunoglobulins, and are present in human serum or plasma. It can bind to antibodies of other species (such as murine antibodies) and interfere with the immunodetection system. Human anti-animal antibodies (HAAA) are the most common xenotropic antibodies. The incidence of HAAA in the population is found to be about 80%, and the causes include contacting animals, receiving monoclonal antibody treatment and the like. In addition, Human anti-mouse antibodies (HAMA) are the most common HAAA. In immunoassay, HAMA binds to murine antibodies, interfering with the immunoassay system, causing experimental outcome bias, known as HAMA effect.

The double antibody sandwich method is exemplified. Normal conditions are as follows: the antigen in the sample binds to both antibodies (capture antibody and labeled antibody), respectively, and the amount of labeled antibody bound to the solid phase is proportional to the concentration of antigen in the sample (FIG. 1 a). False positive: in the absence of antigen, HAMA can bridge the capture antibody and the labeled antibody, thus increasing the amount of binding of the labeled antibody, resulting in false positives (fig. 1 b). False negative: in the presence of antigen, HAMA preferentially binds to the solid phase antibody, creating steric hindrance or a conformational change in the solid phase antibody that renders it unable to bind to the antigen, resulting in false negatives (fig. 1 c).

Disclosure of Invention

The invention mainly aims to provide a kit capable of eliminating HAMA effect, and aims to eliminate HAMA effect in an antigen detection process, reduce the conditions of false positive and false negative, improve the detection accuracy and make the detection process simpler and more convenient.

In order to achieve the above object, the present invention provides a kit for eliminating HAMA effect, comprising a first antibody, a second antibody and a third antibody, wherein the first antibody is a capture antibody specifically binding to an antigen, the second antibody is a humanized antibody against the antigen, the third antibody is a detection antibody against the humanized antibody, and the detection antibody is labeled with a biocatalyst.

Alternatively, the humanized antibody includes any one of a chimeric antibody, a CDR antibody, a resurfaced antibody, a fully human antibody, a single chain antibody, a double chain antibody, and a derivative of the above antibodies.

Alternatively, the heavy chain constant region amino acids of the humanized antibody are human sequences.

Optionally, the detection antibody binds to the heavy chain constant region of the humanized antibody.

Optionally, the biocatalytic enzyme is alkaline phosphatase or horseradish peroxidase.

Optionally, a multi-well plate, a coating buffer, a blocking buffer, a chromogenic substrate and a stop buffer are also included.

The invention provides another kit capable of eliminating HAMA effect, which comprises: the kit comprises a capture antibody for resisting HBeAg, a humanized monoclonal antibody for resisting HBeAg, an anti-humanized monoclonal antibody marked with biological catalytic enzyme, an enzyme label plate, a coating buffer solution, a blocking buffer solution, a chromogenic substrate and a stop solution.

Optionally, the amino acid sequence of the heavy chain constant region of the humanized monoclonal antibody is SEQ ID NO. 1.

Optionally, the amino acid sequence of the heavy chain constant region of the humanized monoclonal antibody is SEQ ID NO. 2.

Optionally, the chromogenic substrate is tetramethylbenzidine or PNPP, and the stop solution is 1M sulfuric acid.

According to the technical scheme, the detection antibody is universally connected with the humanized antibody through the humanized antibody for resisting the antigen and the detection antibody for resisting the humanized antibody with the biological catalytic enzyme label, the humanized antibody for resisting the antigen can eliminate false positive and false negative brought by HAMA effect in the antigen detection process, and the universal detection antibody can reduce the marks of the humanized antibody, so that the antigen detection kit is simpler in preparation method for different antigens, does not need to mark every humanized antibody, can be used for all antigen detection by only using the universal detection antibody for marking, reduces the experimental steps and saves the cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.

FIG. 1 is a schematic diagram of a prior art double antibody sandwich method for detecting antigen, wherein a is a normal case, b is a false positive case, and c is a false negative case.

FIG. 2 is a schematic diagram of antigen detection by the double antibody sandwich method according to an embodiment of the present invention, wherein a is a normal detection condition, and b is a condition that does not affect antigen detection even in the presence of HAMA protein.

FIG. 3 is a graph showing the results of the detection reaction part in example 1 according to the method of the present invention.

FIG. 4 is a graph showing the results of the examination reaction of comparative example 1.

FIG. 5 is a graph showing the results of the examination reaction in comparative example 2.

FIG. 6 is a graph showing the results of the examination reaction in comparative example 3.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a kit capable of eliminating HAMA effect, which comprises a first antibody, a second antibody and a third antibody, wherein the first antibody is a capture antibody specifically binding to an antigen, the second antibody is a humanized antibody against the antigen, the third antibody is a detection antibody against the humanized antibody, and the detection antibody is marked with a biological catalytic enzyme.

Specifically, the first antibody, the second antibody and the third antibody are used only for distinguishing effects, and do not refer to the order thereof. Referring to fig. 2a, a capture antibody and a humanized antibody are respectively combined with different antigenic determinants of an antigen, when the antigen detection kit is used, the capture antibody is coated on a solid phase carrier, a sample to be detected is incubated with the solid phase carrier, so that the antigen is connected with the capture antibody, then the humanized antibody of an anti-antigen is incubated with the solid phase carrier connected with the capture antibody and the antigen, so that the humanized antibody is combined with the antigen, and finally the detection antibody is incubated, so that the detection antibody can be combined with the humanized antibody, and the detection antibody is colored through a mark carried by the detection antibody, thereby indicating the antigen is positive.

According to the technical scheme, by using the humanized antibody for resisting the antigen and the detection antibody with the anti-humanized antibody labeled by the biological catalytic enzyme, as shown in figure 2b, the humanized antibody is combined with the antigen, so that the humanized antibody can not be combined with the HAMA protein, even if the HAMA protein in a sample to be detected can be connected with the capture antibody, the HAMA protein can not be connected with the humanized antibody, and the color development of the detection antibody can not be carried out, so that the condition of false positive is avoided, and the condition of false negative is reduced. The detection antibody is universally connected with the humanized antibody, the humanized antibody of the anti-antigen can eliminate false positive brought by HAMA effect in the antigen detection process, the universal detection antibody can reduce the marks of the humanized antibody, so that the antigen detection kit is simpler in preparation method aiming at different antigen detections, does not need to mark each humanized antibody, can be used in all antigen detections by only using the universal detection antibody for marking, reduces the experimental steps and saves the cost.

Specifically, Alkaline Phosphatase (AP) is an enzyme capable of dephosphorylating a corresponding substrate under Alkaline conditions. PNPP (p-nitro phosphate salt) is a commonly used alkaline phosphatase substrate, and when alkaline phosphatase reacts with PNPP, a yellow water-soluble reaction product is formed, and the amount of the alkaline phosphatase is determined by measuring the absorbance of the reaction product to determine the amount of the reaction product, which absorbs light at λ 405 nm.

Specifically, Horseradish Peroxidase (HRP) is a glycoprotein, and is formed by combining colorless zymoprotein and brown ferriporphyrin. The tetramethyl benzidine is a common horseradish peroxidase substrate, horseradish peroxidase reacts with the tetramethyl benzidine to generate blue precipitate, the blue precipitate is changed into yellow precipitate after the reaction is ended, the reaction solution is yellow, and then the absorbance can be measured at the lambda of 450nm, so that the amount of the reaction product can be judged, and the amount of the horseradish peroxidase can be obtained.

Because the biological catalytic enzymes are used as the markers of the detection antibodies, the number of the detection antibodies can be known according to the number of the biological catalytic enzymes, one detection antibody is connected with one humanized antibody of the anti-antigen, and one humanized antibody is connected with one antigen, so that the number of the antigens can be obtained according to the number of the biological catalytic enzymes, and the positivity and the negativity of the antigens can be judged.

Preferably, the biocatalytic enzyme is horseradish peroxidase, which has high specific activity, stability, small molecular weight and easy preparation of pure enzyme, and is most commonly used.

The invention also provides a method for detecting the antibody coupled with horseradish peroxidase, which comprises the following steps:

(1) dialyzing the detection antibody to be marked into 100mM carbonate buffer solution for 16 h;

(2) activating horseradish peroxidase to oxidize;

(3) mixing the dialyzed detection antibody and the oxidized HRP according to the mass ratio (1:2), and then dialyzing for 16 h;

(4) and (3) terminating the reaction: 4mg NaBH was added₄Standing for 2 h;

(5) and (6) collecting samples.

Specifically, the activation step of horseradish peroxidase specifically comprises the following steps:

20mg of HRP dry powder was weighed and dissolved in 1mL of ddH₂Obtaining an HRP solution in O;

weighing 20mg NaIO₄Dissolved in 1ml of ddH₂Adding all the components into an HRP solution, and stirring and reacting for 10min to obtain an HRP mixed solution;

and sucking 20 mu l of ethylene glycol, adding the ethylene glycol into the HRP mixed solution, stirring for reaction for 10min, and dialyzing for 16h to obtain the oxidized HRP.

Specifically, the sample collection step comprises:

adding saturated ammonium sulfate into the reaction liquid after the reaction is ended according to the volume ratio of 1:1, and standing for 30 min;

centrifuging at 12000rpm at 4 deg.C for 15 min;

discarding the supernatant, and re-dissolving the precipitate with 2mL of 10mM PBS;

centrifuging at 4 deg.C and 12000rpm for 15min, and collecting supernatant;

and storing at 20 ℃ below zero for later use.

Optionally, the kit capable of eliminating HAMA effect further comprises a multi-well plate, a coating buffer solution, a blocking buffer solution, a chromogenic substrate and a stop solution. Specifically, the chromogenic substrate may be PNPP or tetramethylbenzidine. The stop solution is 1M sulfuric acid.

In another aspect, the present invention further provides a kit for eliminating HAMA effect, comprising: the kit comprises a capture antibody for resisting HBeAg, a humanized monoclonal antibody for resisting HBeAg, an anti-humanized monoclonal antibody marked with biological catalytic enzyme, an enzyme label plate, a coating buffer solution, a blocking buffer solution, a chromogenic substrate and a stop solution.

It is understood that the kit is prepared by separately obtaining each component of the kit, labeling the components, and packaging. The capture antibody, the humanized monoclonal antibody and the anti-human monoclonal antibody can be obtained by purchase or self-preparation, and the preparation method of the antibody is not the invention point of the invention, so the preparation method is not described again.

Specifically, the amino acid sequence of the heavy chain constant region of the humanized monoclonal antibody is SEQ ID NO.1, the antibody is of an IgG type, and the coating buffer solution is 0.01M phosphate buffer solution and has the pH value of 7.0; the blocking buffer solution is 0.01M phosphate buffer solution containing 0.05 percent of Tween-20 and pH7.0; the biological catalytic enzyme is alkaline phosphatase or horseradish peroxidase; the chromogenic substrate is tetramethyl benzidine or PNPP, and the stop solution is 1M sulfuric acid.

Optionally, the humanized monoclonal antibody can also be of an IgM type, and the heavy chain constant region amino acid sequence of the humanized monoclonal antibody can be SEQ ID No. 2.

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The antigen detection kit of the embodiment of the invention is prepared from IgG type humanized monoclonal antibody with the heavy chain constant region amino acid sequence of SEQ ID NO.1, detection samples in the embodiment and the comparative example are 50 HBeAg positive samples and 50 HBeAg negative samples, and the detection result accuracy of each kit is analyzed through the detection result.

Example 1 detection of HBeAg by the kit of the present invention

(1) And (6) coating. The capture antibody against HBeAg was diluted with coating buffer, added to the ELISA plate at 100. mu.L per well, and washed three times at 4 ℃ for 24 h.

(2) And (5) sealing. Add blocking solution, add 300. mu.L per well, 37 ℃ for 2h, wash the plate three times.

(3) And (4) antigen reaction. Diluting the sample (containing the antigen HBeAg to be detected), adding the sample into the wells, washing the plate for three times at 37 ℃ for 30min and at 100 mu L per well.

(4) Humanized antibody reaction. The humanized anti-HBeAg monoclonal antibody is diluted, added into the wells, and washed for three times at 37 ℃ for 30min and 100 mu L per well.

(5) And (4) labeled antibody reaction. The antibody obtained from the anti-humanized monoclonal antibody (coupled with HRP) is diluted to a certain multiple, added into each well, and reacted for 30 minutes at 37 ℃ after each well is filled with 100 mu L of the antibody, and plates are washed for three times.

(6) And (4) developing color. Adding tetramethylbenzidine color development solution, 100 μ L per well, keeping away from light, 37 deg.C, and 10 min.

(7) And (6) reading. Stop solution was added at 50. mu.L per well, and absorbance was measured at 450 nm.

Comparative example 1 commercial double-antibody sandwich kit for detecting HBeAg

The HBeAg is detected by a double-antibody sandwich method by using a hepatitis B e antibody ELISA detection kit of Shanghai Kehua bioengineering GmbH as an experimental control. And coating the reaction plate with anti-HBeAg, adding a sample to be detected, and simultaneously adding anti-HBeAg-HRP. If HBeAg exists in the sample to be detected, the HBeAg is combined with the coated anti-HBeAg and the anti-HBeAg-HRP to form a compound. Addition of TMB substrate gave a color reaction and a reading was taken.

Comparative example 2 self-made double-antibody sandwich kit (containing blocker) for detecting HBeAg

The anti-HBeAg capture antibody coats the reaction plate, a sample to be detected which is pretreated by a blocking agent is added, and the blocking agent is a substance which is added into an in-vitro diagnostic reagent system, so that endogenous interference in immunoassay can be eliminated or reduced, and the sensitivity and precision of a kit detection system are improved. anti-HBeAg-HRP was added at the same time. If HBeAg exists in the sample to be detected, the HBeAg is combined with the coated anti-HBeAg and the anti-HBeAg-HRP to form a compound. Addition of TMB substrate gave a color reaction and a reading was taken.

Comparative example 3 detection of HBeAg by a two-antibody Sandwich kit (without blocker)

The anti-HBeAg capture antibody coats the reaction plate, the sample to be detected is added, and the anti-HBeAg-HRP is added at the same time. If the HBeAg exists in the sample to be detected, the HBeAg is combined with the coated anti-HBeAg and the anti-HBeAg-HRP to form a compound, a TMB substrate is added to generate a color reaction, and reading is carried out.

Results of the experiment

The reaction solutions after the detection reactions in example 1 and comparative examples 1 to 3 are shown in FIGS. 3 to 6, respectively, and FIG. 3 to 6 show only 96 reaction wells, respectively, and the OD values of the wells were measured at a dual wavelength of 450/630 using a Thermo Multiskan Sky full-wavelength plate reader. Then, the test results of 50 positive samples and 50 negative samples tested by each kit were counted according to the following method, and the given sample negative or positive was taken as the gold standard, and the counted results were filled in the tables shown in table 1, respectively.

A detection cut-off value (c.o.) was calculated as OD × 0.5 of the blank control well. For this test, the OD values of the blank controls of example 1, comparative example 2 and comparative example 3 are 0.05, 0.07 and 0.07 respectively, and the corresponding critical values are 0.025, 0.035 and 0.035 respectively.

And processing the detection result of the sample. The OD value S/C.O. of the sample is less than or equal to 2.1, and the sample is negative; the OD value S/C.O > 2.1 of the sample is positive.

TABLE 1 statistical tables of results

The reagent box performance calculation formula is as follows. Higher values represent better performance.

The statistical results of the tests of example 1, comparative example 2 and comparative example 3 are shown in tables 2 to 5, respectively. The results of comparing the performance of the different kits are shown in table 6.

TABLE 2 detection results of the inventive method (with humanized monoclonal antibody)

TABLE 3 detection results of the Kehua kit

TABLE 4 self-made kit (containing blocker) test results

TABLE 5 test results from the self-made test kit (without blocker)

TABLE 6 comparison of the Performance of different kits/methods

Kit/method	Sensitivity of the probe	Specificity of	Positive rate of agreement	Negative rate of agreement
					Method of the invention (using humanized monoclonal antibody)	96.0％	92.0％	88.9％	88.5％
Kehua kit	94.0％	88.0％	83.9％	83.0％
					Self-made kit (with blocker)	90.0％	80.0％	75.0％	72.7％
Self-made kit (without blocker)	80.0％	50.0％	53.3％	41.7％

The detection result shows that the humanized monoclonal antibody kit has the highest positive coincidence rate and negative coincidence rate, the minimum probability of false positive and false negative, and the optimal effect.

In the absence of blocking agent, the humanized monoclonal antibody kit has twice the effect of eliminating false positive and false negative compared with the double-antibody sandwich method kit. The main reason for the beneficial effect is that the humanized monoclonal antibody can effectively eliminate the HAAA effect.

The commercial Kowa kit contains a blocking agent, can eliminate partial HAAA effect, and has the effect of reducing false positive and false negative only after that of a humanized monoclonal antibody kit. However, the labeled antibody of the Kowa kit can only bind to HBeAg. When other antigens are detected, recoupling of the antibody is required. The labeled antibody adopted by the humanized monoclonal antibody kit can be combined with the humanized antibody, has universality, and can detect HBeAg, HBsAg, HBcAg and the like.

The above description is only an alternative embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Koron New biological Material Co., Ltd

<120> a kit for eliminating HAMA effect

<130>2020-06-19

<160>2

<170>PatentIn version 3.3

<210>1

<211>330

<212>PRT

<213>Hepatitis B virus

<400>1

Ala Ser Leu Phe Pro Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Leu Pro Ala Leu Phe Pro Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Leu His Gln Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Leu His Gln Lys Ser Cys Asp Lys Leu Phe Pro Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Leu His Gln Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Leu Phe Pro Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Leu Pro Ala Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Leu Pro

260 265 270

Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Phe Pro Ser Pro Gly Lys

325 330

<210>2

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<212>PRT

<213>Hepatitis B virus

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Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn

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Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp

20 25 30

Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp LysTyr Lys Asn Asn Ser

35 40 45

Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys

50 55 60

Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln

65 70 75 80

Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn

85 90 95

Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys

100 105 110

Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg

115 120 125

Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile

130 135 140

Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr

145 150 155 160

Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr

165 170 175

Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln

180 185 190

Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr PheGln Gln

195 200 205

Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val

210 215 220

Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr

225 230 235 240

Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr

245 250 255

Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn

260 265 270

Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala

275 280 285

Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr

290 295 300

Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg

305 310 315 320

Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro

325 330 335

Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu

340 345 350

Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg

355 360 365

Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro

370 375 380

Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val

385 390 395 400

Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala

405 410 415

His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser

420 425 430

Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr

435 440 445

Ala Gly Thr Cys Tyr

450

Claims

1. A kit for eliminating the HAMA effect, comprising a first antibody, a second antibody and a third antibody, wherein the first antibody is a capture antibody specifically binding to an antigen, the second antibody is a humanized antibody against the antigen, the third antibody is a detection antibody against the humanized antibody, and the detection antibody is labeled with a biocatalyst.

2. The kit according to claim 1, wherein the humanized antibody comprises any one of a chimeric antibody, a CDR antibody, a resurfaced antibody, a fully humanized antibody, a single chain antibody, a double chain antibody and a derivative thereof.

3. The kit of claim 2, wherein the amino acids in the heavy chain constant region of the humanized antibody are human.

4. The kit of claim 3, wherein the detection antibody binds to the heavy chain constant region of the humanized antibody.

5. The kit for eliminating HAMA effect according to any one of claims 1 to 4, wherein the biocatalytic enzyme is alkaline phosphatase or horseradish peroxidase.

6. The kit for eliminating HAMA effect of claim 5, further comprising a multi-well plate, a coating buffer, a blocking buffer, a chromogenic substrate and a stop buffer.

7. A kit for eliminating the HAMA effect, comprising: the kit comprises a capture antibody for resisting HBeAg, a humanized monoclonal antibody for resisting HBeAg, an anti-humanized monoclonal antibody marked with biological catalytic enzyme, an enzyme label plate, a coating buffer solution, a blocking buffer solution, a chromogenic substrate and a stop solution.

8. The kit of claim 7, wherein the humanized monoclonal antibody has the amino acid sequence of the heavy chain constant region of SEQ ID No. 1.

9. The kit of claim 7, wherein the humanized monoclonal antibody has the amino acid sequence of the heavy chain constant region of SEQ ID No. 2.

10. The kit for eliminating HAMA effect according to claim 7, wherein the chromogenic substrate is tetramethylbenzidine or PNPP and the stop solution is 1M sulfuric acid.