CN109164263A - A kind of kit and preparation method measuring light chain Kappa concentration - Google Patents
A kind of kit and preparation method measuring light chain Kappa concentration Download PDFInfo
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- CN109164263A CN109164263A CN201811034492.0A CN201811034492A CN109164263A CN 109164263 A CN109164263 A CN 109164263A CN 201811034492 A CN201811034492 A CN 201811034492A CN 109164263 A CN109164263 A CN 109164263A
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Abstract
The present invention relates to field of biotechnology more particularly to a kind of kits and preparation method for measuring light chain Kappa concentration.The technical solution adopted by the present invention is that: a kind of kit measuring Kappa light chain concentration, it is characterized by comprising reagent R1 and reagent R2, wherein it joined biotin and Streptavidin in reagent R2, and biotin combination light chain Kappa antibody ratios are 2:1, Streptavidin combination biotin-goat-anti people's Kappa light chain antibody ratio is 1:1.The invention has the advantages that the kit of measurement Kappa light chain concentration of the invention, reacted using Cascaded amplification, i.e., goat-anti people Kappa light chain antibody connect biotin 1:2, then with Streptavidin 1:1 hybrid reaction, can obviously improve repeatability and sensitivity, the range of linearity can accomplish 0.7-9.0g/L.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of kit for measuring light chain Kappa concentration and preparation sides
Method.
Background technique
Light chain (Light chain, L) is about made of 214 amino acid residues, is typically free of carbohydrate, molecule
Amount is about 24kD.Every light chain is containing there are two the cyclic peptide as composed by intrachain disulfide bond.L chain shares two types: KAPPA (κ)
With Lambda (λ), the type of L chain is always identical on the same native immunoglobulin molecule.In normal human serum κ: λ about
For 2:1.
The paraplasm of cell clone will lead to the increase of monoclonal globulin or immunoglobulin fragment (light chain), this
It will cause the proportional imbalance of κ Yu λ chain, the ratio of κ and λ are not normal predictive of monoclonal Y globulinemia.Generating the case where increasing
Under, the filtration of light chain also increases, and when exceeding the reabsorption ability of renal tubule, which can be combined with quantitative detection and immune ball
Light chain in albumen.
The different types of light chain content of quantitative detection helps to diagnose macroglobulinemia (huge globulin generation increases)
And connective tissue disease (such as rheumatoid arthritis, systemic loupus erythematosus) disease.Infection, acute, chronic hepatitis, cirrhosis etc.
Light chain can also increase in blood, but generally show as κ, λ while increasing；The Urinaries such as nephrosis, diabetes also may occur in which κ, λ simultaneously
Increase.
Existing detection method such as latex intensified exists a large amount of non-specific than the measurement reagent of light chain used in purifying method
Reaction, and the sensitivity and poor repeatability of reagent application, limit its application.Therefore, it should which a kind of new technical side is provided
Case solves the above problems.
Summary of the invention
The object of the present invention is to provide a kind of high sensitivity, a kind of reagent of reproducible measurement light chain Kappa concentration
Box and preparation method.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of kit measuring light chain Kappa concentration, including reagent R1 and reagent R2, each component of the reagent R1 and
Concentration includes:
The each component and concentration of the reagent R2 include:
Further technical solution:
The first buffer solution A and the first buffer solution B in the reagent R1 are PBS buffer solution, HEPES buffer solution, MES buffering
Liquid, Tris buffer, glycine delay the combination of one or more of liquid.
Preservative in the reagent R1 be one of Sodium azide, Proclin-950, Proclin-300, thimerosal or
Several combinations.
The second buffer solution A and the second buffer solution B are PBS buffer solution, HEPES buffer solution, MES buffering in the reagent R2
Liquid, Tris buffer, glycine delay the combination of one or more of liquid.
Stabilizer is the group of one or more of bovine serum albumin(BSA), casein, gelatin, mannitol in the reagent R2
It closes.
Suspending agent in the reagent R2 is glucose, D- trehalose, mannitol, sucrose, maltose, lactose, glycerine
One or more of combination.
Preservative in the reagent R2 is one in Sodium azide, Proclin-950, Proclin-300, thimerosal etc.
Kind.
The R1 reagent pH is between 6.00-8.50, and the R2 reagent pH is between 6.00-8.50.
The biotin combination light chain Kappa antibody ratios are between 1:1-4:1, Streptavidin combination biotin-sheep
Anti-human Kappa light chain antibody ratio is between 0.5:1-4:1.
The preparation method for measuring the kit of Kappa light chain concentration, includes the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept to put into the first buffer solution A while stirring with 0.8-3.2g/L standard, with the mark of 8.75-24.31g/L
Quasi- the first buffer solution B of investment is completely dissolved to material for stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating,
PH value is adjusted between 6.00-8.50, later with 5.8-20.0g/L standard, puts into sodium chloride, after material is completely dissolved again by
According to above-mentioned feeding mode respectively with the standard of 40-120g/L, 0.8%-2.0%, PEG 6000, Proclin-950 are successively put into,
After the completion of feeding intake, continue stirring and be completely dissolved to whole materials for 5-30 minutes, solution is as clear as crystal, Agitation Tank bottom without precipitating,
It is settled to final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, opens magnetic agitation appliance mains switch, adjusts
Stirrer makes solution keep middling speed rotary state, with the standard of 0.5-2.8g/L, it is slow to put into second while stirring to middle-grade revolving speed
Fliud flushing A is put into the second buffer solution B, is careful not to that water is allowed to splash out with the standard of 8.75-24.31g/L, stirs 5-30 minutes to object
Material is completely dissolved, and after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH value between 6.00-8.50, later with 5.8-
20.0g/L standard puts into sodium chloride, according still further to above-mentioned feeding mode respectively with 1-10g/L, 5-30g/ after material is completely dissolved
L, the standard of 0.8-2.0ml/L successively puts into stabilizer, suspending agent and the second preservative, after the completion of feeding intake, continues to stir 5-30
Minute is completely dissolved to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is without precipitating；
Magnetic agitation appliance mains switch is opened on magnetic stirring apparatus with the Standard entertion biotin of 5-20g/L in beaker,
Stirrer is adjusted to middle-grade revolving speed, solution is made to keep middling speed rotary state that sheep is added while stirring with the standard of 25-75ml/L
Anti-human Kappa light chain antibody, stirring 1-4h reaction, the unbonded free antibodies of dialysis removal, are marked after reaction with 5-20g/L
Standard is added Streptavidin and reacts 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's Kappa light chain antibody to be added in Agitation Tank coupling, continues to stir
Mix 5-30 minutes it is as clear as crystal to solution, Agitation Tank bottom is without precipitating.
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified.
Due to the application of above-mentioned technical proposal, the invention has the following advantages over the prior art:
The kit of measurement Kappa light chain concentration of the invention, a molecule Streptavidin can with high degree of specificity with
Four molecular biosciences elements combine, and affinity between the two is extremely strong, form Cascaded amplification reaction.It is reacted using Cascaded amplification,
I.e. goat-anti people Kappa light chain antibody connect biotin 1:2, then with Streptavidin 1:1 hybrid reaction, can obviously improve repeatability
And sensitivity, the range of linearity can accomplish 0.7-9g/L.
Detailed description of the invention
Fig. 1 is in the embodiment of the present invention 4 not using the calibration curve figure of Cascaded amplification reaction R2 reagent.Wherein X-axis indicates
Standard concentration, Y-axis indicate absorbance.
Fig. 2 is in the embodiment of the present invention 4 using the calibration curve figure of Cascaded amplification reaction R2 reagent.Wherein X-axis indicates mark
Quasi- product concentration, Y-axis indicate absorbance.
Specific embodiment
The invention will be further described combined with specific embodiments below:
Embodiment 1:
A kind of kit measuring light chain Kappa concentration, including reagent R1 and reagent R2, each component of the reagent R1 and
Concentration includes the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 0.8g/L, and ten as the first buffer solution B
Two hypophosphite monohydrate disodium hydrogens, concentration 24.31g/L, as the sodium chloride of the first electrolyte, concentration 5.8g/L, as
The PEG 6000 of first macromolecule promotor, concentration 40g/L, and as the Proclin-950 of the first preservative, it is dense
Degree is 2.0%, and each component and concentration of the reagent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A, dense
Degree is 0.5g/L, as the disodium hydrogen phosphate dodecahydrate of the second buffer solution B, concentration 23.4g/L, as the second electrolyte
Sodium chloride, concentration 5.8g/L, as the bovine serum albumin(BSA) of the second stabilizer, concentration 10g/L is helped as second
The D- trehalose of suspension, concentration 5g/L, as the Proclin-950 of the second preservative, concentration 0.8ml/L, and
Goat-anti people Kappa light chain antibody, Streptavidin and biotin, the concentration of this three are respectively 25ml/L, 5g/L and 5g/L.
The preparation method of the kit of the above measurement light chain Kappa concentration, includes the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, investment concentration is 0.8g/L sodium dihydrogen phosphate dihydrate while stirring and concentration is the ten of 24.31g/L
Two hypophosphite monohydrate disodium hydrogens are completely dissolved to material, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjust for stirring 5-30 minutes
Saving pH value is 6.00, the sodium chloride that concentration is 5.8g/L is put into later, after material is completely dissolved, according still further to above-mentioned feeding mode
PEG 6000 and Proclin-950 is successively put into, the concentration of both of the above is respectively 40g/L and 2.0%, after the completion of feeding intake, after
Continuous stirring is completely dissolved for 5-30 minutes to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is settled to final body without precipitating
Product；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, opens magnetic agitation appliance mains switch, adjusts
Stirrer makes solution keep middling speed rotary state, puts into two hypophosphite monohydrates that concentration is 0.5g/L while stirring to middle-grade revolving speed
The disodium hydrogen phosphate dodecahydrate that sodium dihydrogen and concentration are 23.4g/L, is careful not to that water is allowed to splash out, and stirs 5-30 minutes to material
It is completely dissolved, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH value to 8.5, putting into concentration later is 5.8g/L's
Sodium chloride according still further to above-mentioned feeding mode is respectively the standard of 10g/L, 5g/L, 0.8ml/L with concentration after material is completely dissolved
Bovine serum albumin(BSA), D- trehalose and Proclin-950 are successively put into, after the completion of feeding intake, continues to stir 5-30 minutes to whole
Material is completely dissolved, and solution is as clear as crystal, and Agitation Tank bottom is without precipitating；
Magnetic agitation appliance mains switch is opened on magnetic stirring apparatus with the Standard entertion biotin of 5g/L in beaker, is adjusted
Stirrer is saved to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 25ml/L, goat-anti people is added while stirring
Kappa light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 5g/L Standard entertion
Streptavidin reacts 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's Kappa light chain antibody to be added in Agitation Tank coupling, continues to stir
Mix 5-30 minutes it is as clear as crystal to solution, without precipitating, biotin combination light chain Kappa antibody ratios are 2:1, chain for Agitation Tank bottom
Mould Avidin combination biotin-goat-anti people's Kappa light chain antibody ratio is 1:1.
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified.
Embodiment 2
A kind of kit measuring light chain Kappa concentration, including reagent R1 and reagent R2, each component of the reagent R1 and
Concentration includes the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 3.2g/L, and ten as the first buffer solution B
Two hypophosphite monohydrate disodium hydrogens, concentration 8.75g/L, as the sodium chloride of the first electrolyte, concentration 20.0g/L, as
The PEG 6000 of first macromolecule promotor, concentration 80g/L, as the Proclin-300 of the first preservative, concentration is
0.8%, each component and concentration of the reagent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A, and concentration is
2.8g/L, as the disodium hydrogen phosphate dodecahydrate of the second buffer solution B, concentration 11.5g/L, the chlorine as the second electrolyte
Change sodium, concentration 20.0g/L, as the casein of the second stabilizer, concentration 1g/L, the grape as the second suspending agent
Sugar, concentration 30g/L, as the Proclin-300 of the second preservative, concentration 2.0ml/L and goat-anti people Kappa
Light chain antibody, Streptavidin and biotin, the concentration of this three are respectively 75ml/L, 20g/L and 20g/L.
The preparation method of the kit of the above measurement light chain Kappa concentration, includes the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, investment concentration is 3.2g/L sodium dihydrogen phosphate dihydrate while stirring and concentration is the ten of 8.75g/L
Two hypophosphite monohydrate disodium hydrogens are completely dissolved to material, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjust for stirring 5-30 minutes
Saving pH value is 7.00, the sodium chloride that concentration is 20g/L is put into later, after material is completely dissolved, according still further to above-mentioned feeding mode
PEG 6000 and Proclin-300 is successively put into, the concentration of both of the above is respectively 80g/L and 0.8%, after the completion of feeding intake, after
Continuous stirring is completely dissolved for 5-30 minutes to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is settled to final body without precipitating
Product；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, opens magnetic agitation appliance mains switch, adjusts
Stirrer makes solution keep middling speed rotary state, puts into two hypophosphite monohydrates that concentration is 2.8g/L while stirring to middle-grade revolving speed
The disodium hydrogen phosphate dodecahydrate that sodium dihydrogen and concentration are 11.5g/L, is careful not to that water is allowed to splash out, and stirs 5-30 minutes to material
It is completely dissolved, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH value to 7, put into the chlorine that concentration is 20.0g/L later
Change sodium, after material is completely dissolved according still further to above-mentioned feeding mode respectively with concentration for 1g/L, 30g/L, 2.0ml/L standard according to
Secondary investment casein, glucose and Proclin-300, after the completion of feeding intake, continue stirring 5-30 minutes it is completely molten to whole materials
Solution, solution is as clear as crystal, and Agitation Tank bottom is without precipitating；
Magnetic agitation appliance mains switch is opened on magnetic stirring apparatus with the Standard entertion biotin of 20g/L in beaker, is adjusted
Stirrer is saved to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 75ml/L, goat-anti people is added while stirring
Kappa light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 20g/L Standard entertion
Streptavidin reacts 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's Kappa light chain antibody to be added in Agitation Tank coupling, continues to stir
Mix 5-30 minutes it is as clear as crystal to solution, without precipitating, biotin combination light chain Kappa antibody ratios are 2:1, chain for Agitation Tank bottom
Mould Avidin combination biotin-goat-anti people's Kappa light chain antibody ratio is 1:1.
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified.
Embodiment 3
A kind of kit measuring light chain Kappa concentration, including reagent R1 and reagent R2, each component of the reagent R1 and
Concentration includes the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 2g/L, and 12 as the first buffer solution B
Hypophosphite monohydrate disodium hydrogen, concentration 16g/L, as the sodium chloride of the first electrolyte, concentration 12g/L is high as first
The PEG 6000 of molecule promotor, concentration 120g/L, as the thimerosal of the first preservative, concentration 1%, the examination
The each component and concentration of agent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A, concentration 1.5g/L, as
The disodium hydrogen phosphate dodecahydrate of two buffer solution Bs, concentration 17g/L, as the sodium chloride of the second electrolyte, concentration is
13g/L, as the gelatin of the second stabilizer, concentration 5g/L, as the maltose of the second suspending agent, concentration 18g/L,
As the thimerosal of the second preservative, concentration 1.3ml/L and goat-anti people Kappa light chain antibody, Streptavidin and biology
Element, the concentration of this three are respectively 50ml/L, 12/L and 12g/L.
The preparation method of the kit of the above measurement light chain Kappa concentration, includes the following steps,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, makes solution
Middling speed rotary state is kept, puts into the Shi Ershui that concentration is 2g/L sodium dihydrogen phosphate dihydrate and concentration is 16g/L while stirring
Disodium hydrogen phosphate is closed, is completely dissolved to material within stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH
Value is 6.00, puts into the sodium chloride that concentration is 12g/L later, after material is completely dissolved, successively according still further to above-mentioned feeding mode
PEG 6000 and thimerosal are put into, the concentration of both of the above is respectively 120g/L and 1%, after the completion of feeding intake, continues to stir 5-30
Minute is completely dissolved to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is settled to final volume without precipitating；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified；
B), prepared by reagent R2；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, opens magnetic agitation appliance mains switch, adjusts
Stirrer makes solution keep middling speed rotary state, puts into two hypophosphite monohydrates that concentration is 1.5g/L while stirring to middle-grade revolving speed
The disodium hydrogen phosphate dodecahydrate that sodium dihydrogen and concentration are 17g/L, is careful not to that water is allowed to splash out, and stirring 5-30 minutes complete to material
Fully dissolved after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH value to 6, puts into the chlorination that concentration is 13g/L later
Sodium, after material is completely dissolved according still further to above-mentioned feeding mode respectively with concentration for 5g/L, 18g/L, 1.3ml/L standard successively
Gelatin, maltose and thimerosal are put into, after the completion of feeding intake, continues stirring and is completely dissolved to whole materials for 5-30 minutes, solution is clear
Clear transparent, Agitation Tank bottom is without precipitating；
Magnetic agitation appliance mains switch is opened on magnetic stirring apparatus with the Standard entertion biotin of 12g/L in beaker, is adjusted
Stirrer is saved to middle-grade revolving speed, so that solution is kept middling speed rotary state, with the standard of 50ml/L, goat-anti people is added while stirring
Kappa light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 12g/L Standard entertion
Streptavidin reacts 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's Kappa light chain antibody to be added in Agitation Tank coupling, continues to stir
Mix 5-30 minutes it is as clear as crystal to solution, Agitation Tank bottom is without precipitating.Biotin combination light chain Kappa antibody ratios are 2:1, chain
Mould Avidin combination biotin-goat-anti people's Kappa light chain antibody ratio is 1:1.
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored
In finished pot, it is identified.
Embodiment 4
A kind of application for the kit for measuring light chain Kappa concentration of the present invention, its working principle is that: a molecule strepto- is affine
Element can be with high degree of specificity in conjunction with four molecular biosciences elements, and affinity between the two is extremely strong, and it is anti-to form Cascaded amplification
It answers.Light chain Kappa in sample and Streptavidin-biotin in reagent-anti human light chain Kappa antibody phase in the solution
It meets, is reacted using Cascaded amplification, form antigen-antibody complex, be aggregated, generate turbidity variation, cause the increasing of absorbance
Add.Content of absorbance in the presence of sufficient antibodies with light chain Kappa in sample caused by the turbidity changes is positively correlated,
Under 340nm wavelength, by compared with the calibration object of known concentration, can quantitative detection go out the content of light chain Kappa in sample.
The kit for not using Cascaded amplification to react using R2 reagent, is calibrated using standard items, as a result such as 1 institute of table
Show, calibration curve is as shown in Figure 1.
Table 1, R2 reagent do not use Cascaded amplification to react the calibration results:
| Calibration object concentration (g/L) | DOD (absorbance) |
| 0.00 | 0.0101 |
| 1.14 | 0.0660 |
| 2.28 | 0.1339 |
| 4.56 | 0.2403 |
| 9.12 | 0.2571 |
As a result parse: as shown in figure 1 and table 1, there is hook effect when being greater than 4.56g/L in calibration object concentration.
R2 reagent uses the kit of Cascaded amplification reaction, is calibrated using standard items, and the results are shown in Table 2, calibration
Curve is as shown in Figure 2.
Table 2, R2 reagent react the calibration results using Cascaded amplification:
As a result parse: as shown in Fig. 2 and table 2, the range of linearity can accomplish 0.7-9.0g/L.
Embodiment 5
Precision CV detection:
The kit that R2 reagent does not use Cascaded amplification to react, takes 3 part of one clinical serum sample at random, using full-automatic raw
Change analyzer and detection 10 times is repeated to same sample, testing result is as shown in table 3.
Table 3: the Kappa light chain content (10 times) and the coefficient of variation of sample are detected
As a result it parses: precision CV > 5%.
R2 reagent uses the kit of Cascaded amplification reaction, takes 4 parts of clinical serum samples at random, uses full-automatic biochemical point
Analyzer repeats detection 10 times to same sample, and testing result is as shown in table 4.
Table 4: the Kappa light chain content (10 times) and the coefficient of variation of sample are detected
As a result it parses: precision CV < 4%.
Conclusion: being reacted using Cascaded amplification, i.e., 1 part of goat-anti people's Kappa light chain antibody connects 2 parts of biotin, then with 1
The Streptavidin hybrid reaction of part, can obviously improve repeatability and sensitivity, the range of linearity can accomplish 0.7-9.0g/L.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements or replace, these are improved or replacement
It should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of kit for measuring light chain Kappa concentration, it is characterised in that: including reagent R1 and reagent R2, the reagent R1
Each component and concentration include:
The each component and concentration of the reagent R2 include:
2. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the reagent R1
It is PBS buffer solution, HEPES with the first buffer solution A, the first buffer solution B, the second buffer solution A and the second buffer solution B in R2
Buffer, MES buffer, Tris buffer, glycine delay the combination of one or more of liquid.
3. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the reagent R1
It is the combination of one or more of Sodium azide, Proclin-950, Proclin-300, thimerosal with the preservative in R2.
4. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the reagent R2
Middle stabilizer is the combination of one or more of bovine serum albumin(BSA), casein, gelatin, mannitol.
5. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the reagent R2
In suspending agent be one or more of glucose, D- trehalose, mannitol, sucrose, maltose, lactose, glycerine group
It closes.
6. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the reagent R1,
The pH of R2 is between 6.00-8.50.
7. a kind of kit for measuring light chain Kappa concentration according to claim 1, it is characterised in that: the biotin
In conjunction with light chain Kappa antibody ratios between 1:1-4:1, Streptavidin combination biotin-goat-anti people Kappa light chain antibody ratio
Example is between 0.5:1-4:1.
8. a kind of kit for measuring light chain Kappa concentration according to claim 2, it is characterised in that: described first is slow
Fliud flushing A and the second buffer solution A are sodium dihydrogen phosphate dihydrate, and the first buffer solution B and the second buffer solution B are 12 hypophosphite monohydrate hydrogen
Disodium.
9. the preparation method of the kit such as any measurement light chain Kappa concentration of claim 1-8, it is characterised in that: including such as
Lower step,
A), prepared by reagent R1；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, adjusting stirrer to middle-grade revolving speed, keeps solution
Middling speed rotary state puts into the first buffer solution A with 0.8-3.2g/L standard while stirring, is thrown with the standard of 8.75-24.31g/L
Enter the first buffer solution B, is completely dissolved to material within stirring 5-30 minutes, after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts
PH value, later with 5.8-20.0g/L standard, puts into sodium chloride, according still further to upper after material is completely dissolved between 6.00-8.50
Feeding mode is stated respectively with the standard of 40-120g/L, 0.8%-2.0%, PEG 6000, Proclin-950 is successively put into, feeds intake
After the completion, continue stirring to be completely dissolved to whole materials for 5-30 minutes, solution is as clear as crystal, and Agitation Tank bottom is without precipitating, constant volume
To final volume；
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored in into
In product tank, it is identified；
B), prepared by reagent R2；
Appropriate purified water is first added in Agitation Tank on magnetic stirring apparatus, opens magnetic agitation appliance mains switch, adjusts stirring
Son makes solution that middling speed rotary state be kept to put into the second buffer while stirring with the standard of 0.5-2.8g/L to middle-grade revolving speed
A is put into the second buffer solution B, is careful not to that water is allowed to splash out with the standard of 8.75-24.31g/L, and stirring 5-30 minutes complete to material
Fully dissolved after the as clear as crystal Agitation Tank bottom of solution is without precipitating, adjusts pH value between 6.00-8.50, later with 5.8-
20.0g/L standard puts into sodium chloride, according still further to above-mentioned feeding mode respectively with 1-10g/L, 5-30g/ after material is completely dissolved
L, the standard of 0.8-2.0ml/L successively puts into stabilizer, suspending agent and the second preservative, after the completion of feeding intake, continues to stir 5-30
Minute is completely dissolved to whole materials, and solution is as clear as crystal, and Agitation Tank bottom is without precipitating；
Magnetic agitation appliance mains switch is opened on magnetic stirring apparatus with the Standard entertion biotin of 5-20g/L in beaker, is adjusted
Stirrer makes solution keep middling speed rotary state, with the standard of 25-75ml/L, goat-anti people is added while stirring to middle-grade revolving speed
Kappa light chain antibody, stirring 1-4h reaction, the unbonded free antibodies of dialysis removal, are added after reaction with 5-20g/L standard
Enter Streptavidin reaction 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's Kappa light chain antibody to be added in Agitation Tank coupling, continues to stir 5-
30 minutes as clear as crystal to solution, and Agitation Tank bottom is without precipitating.
What is dissolved matches liquid according to millipore filter operating instruction, and by filtering with microporous membrane, filtered solution is stored in into
In product tank, it is identified.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568180A (en) * | 2019-09-12 | 2019-12-13 | 苏州普瑞斯生物科技有限公司 | Glycocholic acid detection kit and preparation method thereof |
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