CN109164262A - A kind of kit and preparation method measuring free light chain Lambda concentration - Google Patents
A kind of kit and preparation method measuring free light chain Lambda concentration Download PDFInfo
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- CN109164262A CN109164262A CN201811034491.6A CN201811034491A CN109164262A CN 109164262 A CN109164262 A CN 109164262A CN 201811034491 A CN201811034491 A CN 201811034491A CN 109164262 A CN109164262 A CN 109164262A
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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 free light chain Lambda concentration.The technical solution adopted by the present invention is that: a kind of kit measuring Lambda free 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 free light chain lambda antibody ratios are 2:1, Streptavidin combination biotin-goat-anti people's lambda free light chain antibody ratios are 1:1.The invention has the advantages that the kit of measurement Lambda free light chain concentration of the invention, it is reacted using Cascaded amplification, i.e. goat-anti people lambda free light chain antibody connects biotin 1:2, again with Streptavidin 1:1 hybrid reaction, can obviously improve repeatability and sensitivity, the range of linearity can accomplish 4-140mg/L.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of kits and system for measuring free light chain Lambda concentration
Preparation Method.
Background technique
Immunoglobulin (Ig) light chain is divided into 2 types of κ (kappa) and λ (lambda), on each Ig molecule only one
The ratio of the light chain of type, mankind κ (kappa) and λ (lambda) are 6: 4.Light chain is that can pass freely through glomerular basement membrane
Small protein is returned in blood circulation in renal tubule by reabsorption, so only a small amount of light chain exists in normal human urine.Work as hair
When raw metabolism disorder and Huppert's disease, it will appear a large amount of free light chains in blood, and by being discharged in urine, as Bence Jones protein
(Bence-Jones albumen).Normal human serum free light chain concentration is 3~19mg/L, and wherein λ type free light chain is 6~26mg/
L, κ/λ ratio are 0.26~1.65.The κ type free light chain of abnormal conditions and with κ/λ ratio or exception λ type free light chain and
Situations such as κ/λ ratio or simultaneously three are abnormal, to the quick of non-secretory myeloma (nonsecretory myeloma, NSM)
Perception is 65%~70%, and the sensibility of disease (monoclonal gammopathies, MGP) is 88%~98%；Facilitate list
The early diagnosis of cloned light chain disease, AL- amyloidosis, it can also be used to whether be recurred after chemotherapy or itself autologous peripheral blood stemcell transplant
Monitoring, if chemotherapy or transplantation effect are good, sufferer serum κ type free light chain, λ type free light chain and κ/λ ratio exist
Normal level or compared to having clear improvement before treatment.Measurement for free light chain, it is domestic mainly to pass through latex intensified ratio at present
Purifying method measures Lambda free light chain；But in clinical application, the Lambda free light chain of Freelite series measures reagent
There are a large amount of non-specific responding, and the sensitivity and poor repeatability of reagent 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, the examination of reproducible measurement free light chain Lambda concentration
Agent box and preparation method.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of kit measuring free light chain Lambda concentration, including reagent R1 and reagent R2, the reagent R1's is each
Component and concentration include:
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.
For the biotin combination free light chain lambda antibody ratios between 1:1-4:1, Streptavidin combines biology
Element-goat-anti people's lambda free light chain antibody ratios are between 0.5:1-4:1.
The preparation method for measuring the kit of free light chain Lambda 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 75-150ml/L
Anti-human lambda free light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 5-
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 lambda free light chain antibody to be added in Agitation Tank coupling, after
Continuous stirring 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 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 free light chain Lambda concentration of the invention, a molecule Streptavidin can be with high degree of specificity
In conjunction with four molecular biosciences elements, affinity between the two is extremely strong on ground, forms Cascaded amplification reaction, anti-using Cascaded amplification
Answer, i.e., goat-anti people lambda free light chain antibody connect biotin 1:2, then with Streptavidin 1:1 hybrid reaction, can obviously change
Kind repeatability and sensitivity, the range of linearity can accomplish 4-140mg/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 free light chain Lambda concentration, including reagent R1 and reagent R2, the reagent R1's is each
Component and concentration include the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 0.8g/L, as the first buffer
The disodium hydrogen phosphate dodecahydrate of B, concentration 24.31g/L, as the sodium chloride of the first electrolyte, concentration 5.8g/L,
As the PEG 6000 of the first macromolecule promotor, concentration 40g/L, and as the Proclin-950 of the first preservative,
Its concentration is 2.0%, and each component and concentration of the reagent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A,
Its concentration is 0.5g/L, as the disodium hydrogen phosphate dodecahydrate of the second buffer solution B, concentration 23.4g/L, as the second electricity
Solve the sodium chloride of matter, concentration 5.8g/L, as the bovine serum albumin(BSA) of the second stabilizer, concentration 10g/L, as the
The D- trehalose of two suspending agents, concentration 5g/L, as the Proclin-950 of the second preservative, concentration 0.8ml/L,
And goat-anti people's lambda free light chain antibody, Streptavidin and biotin, the concentration of this three are respectively 75ml/L, 5g/L
And 5g/L.
The preparation method of the kit of the above measurement Lambda free 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, 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 75ml/L, goat-anti people is added while stirring
Lambda free light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 5g/L standard
Streptavidin is added and reacts 1-4h, after reaction the unbonded Biotin-Antibody of dialysis removal；
Finally there is Streptavidin-biotin-goat-anti people's lambda free light chain antibody to be added in Agitation Tank coupling, after
Continuous stirring 5-30 minutes as clear as crystal to solution, and Agitation Tank bottom is without precipitating, biotin combination free light chain lambda antibody ratio
Example is 2:1, and Streptavidin combination biotin-goat-anti people's lambda free light chain antibody ratios are 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 free light chain Lambda concentration, including reagent R1 and reagent R2, the reagent R1's is each
Component and concentration include the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 3.2g/L, as the first buffer
The disodium hydrogen phosphate dodecahydrate of B, concentration 8.75g/L, as the sodium chloride of the first electrolyte, concentration 20.0g/L,
It is dense as the Proclin-300 of the first preservative as the PEG 6000 of the first macromolecule promotor, concentration 80g/L
Degree is 0.8%, and each component and concentration of the reagent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A, dense
Degree is 2.8g/L, as the disodium hydrogen phosphate dodecahydrate of the second buffer solution B, concentration 11.5g/L, as the second electrolyte
Sodium chloride, concentration 20.0g/L, as the casein of the second stabilizer, concentration 1g/L, as the second suspending agent
Glucose, concentration 30g/L, as the Proclin-300 of the second preservative, concentration 2.0ml/L and goat-anti people
Lambda free light chain antibody, Streptavidin and biotin, the concentration of this three are respectively 150ml/L, 20g/L and 20g/L.
The preparation method of the kit of the above measurement Lambda free 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, 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 150ml/L, goat-anti people is added while stirring
Lambda free light chain antibody, stirring 1-4h reaction, the unbonded free antibodies of dialysis removal, are marked after reaction with 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 lambda free light chain antibody to be added in Agitation Tank coupling, after
Continuous stirring 5-30 minutes as clear as crystal to solution, and Agitation Tank bottom is without precipitating, biotin combination free light chain lambda antibody ratio
Example is 2:1, and Streptavidin combination biotin-goat-anti people's lambda free light chain antibody ratios are 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 free light chain Lambda concentration, including reagent R1 and reagent R2, the reagent R1's is each
Component and concentration include the sodium dihydrogen phosphate dihydrate as the first buffer solution A, concentration 2g/L, as the first buffer solution B
Disodium hydrogen phosphate dodecahydrate, concentration 16g/L, as the sodium chloride of the first electrolyte, concentration 12g/L, as
The PEG 6000 of first macromolecule promotor, concentration 120g/L, as the thimerosal of the first preservative, concentration 1%,
The each component and concentration of the reagent R2 include the sodium dihydrogen phosphate dihydrate as the second buffer solution A, concentration 1.5g/L,
As the disodium hydrogen phosphate dodecahydrate of the second buffer solution B, concentration 17g/L is dense as the sodium chloride of the second electrolyte
Degree is 13g/L, and as the gelatin of the second stabilizer, concentration 5g/L, as the maltose of the second suspending agent, concentration is
18g/L, as the thimerosal of the second preservative, concentration 1.3ml/L and goat-anti people's lambda free light chain antibody, strepto-
Avidin and biotin, the concentration of this three are respectively 115ml/L, 12/L and 12g/L.
The preparation method of the kit of the above measurement free light chain Lambda 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 115ml/L, goat-anti people is added while stirring
Lambda free light chain antibody, stirring 1-4h reaction, the unbonded free antibodies of dialysis removal, are marked after reaction with 12g/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 lambda free light chain antibody to be added in Agitation Tank coupling, after
Continuous stirring 5-30 minutes as clear as crystal to solution, and Agitation Tank bottom is without precipitating.Biotin combination free light chain lambda antibody ratio
Example is 2:1, and Streptavidin combination biotin-goat-anti people's lambda free light chain antibody ratios are 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 free light chain Lambda concentration of the present invention, its working principle is that: a strand
Mould Avidin can be with high degree of specificity in conjunction with four molecular biosciences elements, and affinity between the two is extremely strong, form cascade
Iodine.The anti-human free light chain λ antibody of Streptavidin-biotin-in free light chain λ and reagent in sample is in solution
In meet, reacted using Cascaded amplification, formed antigen-antibody complex, be aggregated, generate turbidity variation, cause absorbance
Increase.Content of absorbance in the presence of sufficient antibodies with free light chain λ 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 free light chain λ 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 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 | -0.0030 |
| 9.41 | 0.0869 |
| 27.28 | 0.1471 |
| 76.66 | 0.3025 |
| 140.84 | 0.2633 |
As a result parse: as shown in figure 1 and table 1, there is hook effect when being greater than 76.66g/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:
| Calibration object concentration (g/L) | DOD (absorbance) |
| 0 | -0.0012 |
| 9.41 | 0.0981 |
| 27.28 | 0.2505 |
| 76.66 | 0.5711 |
| 140.84 | 0.8694 |
As a result parse: as shown in Fig. 2 and table 2, the range of linearity can accomplish 4-140g/L.
Embodiment 5
Precision CV detection:
The kit that R2 reagent does not use Cascaded amplification to react, takes 3 parts of clinical serum samples at random, uses full-automatic biochemical
Analyzer repeats detection 10 times to same sample, and testing result is as shown in table 3.
Table 3: the Lambda free light chain content (10 times) and the coefficient of variation of sample are detected
| Serial number | Sample 1 (g/L) | Sample 2 (g/L) | Sample 3 (g/L) |
| 1 | 12.24 | 65.76 | 127.00 |
| 2 | 12.47 | 64.49 | 91.62 |
| 3 | 12.25 | 68.83 | 118.40 |
| 4 | 11.55 | 65.65 | 98.30 |
| 5 | 12.29 | 64.43 | 115.96 |
| 6 | 12.84 | 69.52 | 111.36 |
| 7 | 13.02 | 66.47 | 109.81 |
| 8 | 11.89 | 64.72 | 127.80 |
| 9 | 12.54 | 70.06 | 129.24 |
| 10 | 11.67 | 68.50 | 100.43 |
| Mean value | 12.28 | 66.84 | 112.99 |
| SD | 0.47 | 2.18 | 13.17 |
| CV | 3.86% | 3.26% | 11.65% |
As a result it parses: precision CV > 3%.
R2 reagent uses the kit of Cascaded amplification reaction, takes 3 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 Lambda free light chain content (10 times) and the coefficient of variation of sample are detected
As a result it parses: precision CV < 2%.
Conclusion: being reacted using Cascaded amplification, i.e., 1 part of goat-anti people's lambda free light chain antibody connects 2 parts of biotin,
Streptavidin hybrid reaction with 1 part again, can obviously improve repeatability and sensitivity, the range of linearity can accomplish 4-140mg/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 (10)
1. a kind of kit for measuring free light chain Lambda concentration, it is characterised in that: including reagent R1 and reagent R2, the examination
The each component and concentration of agent R1 include:
The each component and concentration of the reagent R2 include:
2. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the examination
The first buffer solution A, the first buffer solution B, the second buffer solution A and the second buffer solution B in agent R1 and R2 be PBS buffer solution,
HEPES buffer solution, MES buffer, Tris buffer, glycine delay the combination of one or more of liquid.
3. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the examination
Preservative in agent R1 is the combination of one or more of Sodium azide, Proclin-950, Proclin-300, thimerosal.
4. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: described the
One buffer solution 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 hydration phosphorus
Sour disodium hydrogen.
5. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the examination
Stabilizer is the combination of one or more of bovine serum albumin(BSA), casein, gelatin, mannitol in agent R2.
6. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the examination
Suspending agent in agent R2 is one or more of glucose, D- trehalose, mannitol, sucrose, maltose, lactose, glycerine
Combination.
7. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the examination
Preservative in agent R2 is one of Sodium azide, Proclin-950, Proclin-300, thimerosal etc..
8. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the R1
Reagent pH is between 6.00-8.50, and the R2 reagent pH is between 6.00-8.50.
9. a kind of kit for measuring free light chain Lambda concentration according to claim 1, it is characterised in that: the life
Object element combination free light chain lambda antibody ratios are between 1:1-4:1, Streptavidin combination biotin-goat-anti people lambda
Free light chain antibody ratios are between 0.5:1-4:1.
10. the preparation method of the kit such as any measurement free light chain Lambda concentration of claim 1-9, it is characterised in that:
Include 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, 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 75-150ml/L, goat-anti people is added while stirring to middle-grade revolving speed
Lambda free light chain antibody, stirring 1-4h reaction, the free antibodies that dialysis removal is not associated with after reaction, with 5-20g/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 lambda free 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 into
In product tank, it is identified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811034491.6A CN109164262A (en) | 2018-09-05 | 2018-09-05 | A kind of kit and preparation method measuring free light chain Lambda concentration |
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| CN201811034491.6A CN109164262A (en) | 2018-09-05 | 2018-09-05 | A kind of kit and preparation method measuring free light chain Lambda concentration |
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| CN201811034491.6A Pending CN109164262A (en) | 2018-09-05 | 2018-09-05 | A kind of kit and preparation method measuring free light chain Lambda concentration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113092786A (en) * | 2021-04-09 | 2021-07-09 | 北京美联泰科生物技术有限公司 | Buffer solution and application thereof in central nervous system specific protein detection kit |
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