CN1427257A - Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying high density lipoprotein content in human urine and its preparation method - Google Patents
Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying high density lipoprotein content in human urine and its preparation method Download PDFInfo
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Abstract
An enzyme-linked immunosorbent assay kit for detecting the content of high-density lipoprotein in human urine and its preparing process are disclosed. It can be used to diagnose the nephrosis. Its advantages are high speed, specificity, and sensitivity and simple method.
Description
Technical field
The present invention relates to biological technology products, be specifically related to the enzyme-linked immunosorbent assay (ELISA) reagent box and the preparation method of low-density lipoprotein (High density lipoprotein HDL) content in a kind of people's urine.
Background technology
Increasing to the test item of range protein in kidney patient and the diabetic's urine at present, but the HDL Determination on content is not appeared in the newspapers so far in the urine.
Summary of the invention
Technical matters to be solved by this invention is to overcome the weak point of detection method in the past, develops the diagnostic kit of a kind of high sensitivity and high specific.
The invention provides a kind of HDL content enzyme and exempt from kit, this kit is with antibody HDL (IgG) bag quilt, and antibody HDL (IgG) connects horseradish peroxidase for detecting the sandwich method of antibody, detects the HDL content in people's urine.And form by following reagent:
1. the HDL IgG antibody is wrapped in advance by 1 of plate
2. HDL IgG antibody enzyme labeling liquid is 1 bottle
3. HDL standard (2500,1250,625,56,10,0ng/ml) is each 1 bottle
4. concentrated cleaning solution is 1 bottle
5. substrate solution is towards each 1 bottle of liquid first, second
6. stop buffer is 1 bottle
Another technical matters to be solved of the present invention has provided the preparation method of the ELISA kit of HDL content in the above-mentioned urine, this method comprises the following steps: one, starting material and specification normal human serum animal thereof: new zealand white rabbit, healthy male, body weight 2～3kg ultracentrifuge: the board 80-P-7 of Hitachi type, RPZ-48T district band rotor, the RPS-50-2 level
The rotor density gradient forms instrument, Hitachi's board, DGP-2 type Unic ultraviolet spectrometry degree meter: SP-800 type enzyme-linked immunoassay instrument: Labosystems Dragon Multiskan MK3UV754 spectrophotometer vortex mixer, XW-80 type PHS-20 type precision acidity meter electrophoresis apparatus (Shanghai) ELISA Plate, the ELISA of East China University of Science measures CV% (%)＜10% horseradish peroxidase (RZ3.0, SigMA) NaIO
4A.R (import packing) NaBH
4Other reagent Ns of A.R (import packing) a
2HPO
412H
2O A.R citric acid A.RNacl A.R glycerine chemical pure H
2O
2A.RTween-20 chemical pure o-phenylenediamine chemical pure H
2SO
4A.R distilled water must meet " the regulation High Density lipoprotein of Chinese pharmacopoeia (1990), HDL standard items (SigMA) two, coated antibody HDL Polyclonal Antibody Preparation () district band density gradient centrifugation purifying HDL operating process:
This paper is with density with reference to Hinton (1976) district, but has done bigger improvement.Use the 30-P-7 of Hitachi hydro-extractor, the RPZ-48T rotor is done band centrifugation.Use earlier Abbe refractometer, it is 1.4 liquid that NaBr gradient liquid is mixed with density, is that 1.4 NaBr and density are that 1.0 distilled water forms instrument through density gradient and added in district's band rotor by lateral opening then with density, and rotating speed maintains 2800/rpm at this moment.When adding to mesopore and having gradient liquid to flow out, to inject density with constant flow pump be 1.4 human serum 50ml through lateral opening again.After treating that blood sample all adds, rotating speed is risen to 42000/rpm obtain four peaks after centrifugal 18 hours altogether, receive sample by mesopore, sample flow records four lipoprotein component peaks through the Unic ultraviolet spectrophotometer.Wherein first peak is creamy white, and concentrates on first pipe, is very low density lipoprotein (VLDL) (vLDL).All the other several peaks all are light yellow, are respectively low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (HDL).All the other are degrease serum, contain other foreign protein peaks.With the peak of collecting, select the main peak few top pipe can not be too wide, in order to avoid sneak into intermediated-density lipoprotein (IDL).(2) preparation of lipoprotein antiserum HDL
Prepare protein Antiserum Preparation method routinely.At first the HDL with purifying is dissolved in the 0.01MPH7.4 phosphate buffer, add the Fu Shi Freund's complete adjuvant emulsification of equivalent after, give new zealand white rabbit, injection 2.0mg is fundamental immunity in nape portion multiple spot and the foot pad.Per two weeks are strengthened once, and immunity is 6 times altogether.The blood drawing test in 8-10 days of last immunity back is tired, and the satisfied person that tires collects antiserum by the heart blood drawing, and antiserum is again through ammonium sulfate (the 50% and 33% saturated (NH that saltouts for twice
4) SO
4) the fractional precipitation purifying becomes the anti-people of rabbit (HDL) IgG antibody (be called for short HDL IgG antibody).(3) preparation of the anti-people HDL of enzyme mark rabbit IgG antibody cross-linking agent.
The crosslinked acquisition of sodium periodate method " enzyme labelled antibody IgG " of improvement of the rabbit of purified gained anti-people HDL IgG antibody and horseradish peroxidase.Reagent preparation and operation steps are as follows: 1. sodium periodate labelling method reagent (1) 0.06M NaIO
4(0.13 gram NaIO
4, adding water to 10ml) and (2) 0.16M ethylene glycol solution 0.1ml adds water to 10ml (3) NaBH
4(5mg/ml, NaBH
45mg adds water to 1ml) (4) 0.05M PH9.5 carbonate buffer solution
First liquid: natrium carbonicum calcinatum 10.6 grams add water to 500ml
Second liquid: anhydrous sodium bicarbonate 16.8 grams add water to 1000ml
Get first liquid 16ml+ second liquid 34ml, add water to the 2. operation steps of periodic acid labelling method of 200ml (5) 0.02M PH7.4 PBS (with 0.1M PH7.4 PBS dilution)
7.5mg HRP+0.5ml dissolved in distilled water
↓
Add 0.06M NaIO
40.5ml, mix rearmounted 4 ℃, 30 minutes
↓
Add 0.16M ethylene glycol 0.5ml room temperature 30 minutes
↓
The rabbit lipotropism protein antibodies HDL IgG 0.5ml that adds mg/ml
(containing HDL IgG antibody 7mg approximately), mix back dress bag filter, use 0.05M, the pH9.6 carbonic acid buffer liquid of dialysing
↓
Sucking-off next day dialysate adds NaBH
4Solution 0.2ml (5mg/ml) put refrigerator 2 hours
↓
The above-mentioned bond mixed liquor of sucking-off adds the equal-volume saturated ammonium sulfate, behind 4 ℃ 30 ' in the refrigerator, abandons supernatant in centrifugal 4000 rev/mins * 15 minutes, and precipitation is dissolved in the liquid of dialysing among the 1ml PH7.4 0.02M PBS, changes liquid three times, each 1000ml
↓
Next day, the centrifugal again insolubles of removing promptly gets " enzyme labelled antibody IgG ", be sub-packed in the ampoule sealing after, put-40 ℃ of behaviour that preserve (four) lipoprotein HDL enzyme linked immunosorbent assay (ELISA) double antibody sandwich methods
Make the step urine sample: the diluted liquid of stoste bag: Na
2CO
30.16 gram, NaHCO
32.9 gram, NaN
30.02 gram adds water
Become the pH9.6 carbonate buffer solution to 100ml.Sample diluting liquid: NaCL 8 grams, KH
2PO
40.2 gram, Na
2HPO
42.9 gram, KCL0.2
Gram, T ween-20 0.5ml, NaN
30.2 gram adds water to 1000ml,
Add the 10ml calf serum with preceding every 100ml and become pH7.4PBS-Tween
20 sample diluting liquids.Cleansing solution: Tris 2.42g, 1N HCL 13ml, Tween-20 0.5ml adds water to 1000ml
Become pH7.4 0.02M Tris-Tween 20 cleansing solutions.Matrix liquid: 0.1M Na
2HPO
45.14ml (3.6 gram Na
2HPO
4Add water 100ml), 0.05M
Citric acid 4.86ml (1 gram citric acid adds water 100ml) o-phenylenediamine 4mg, 3%
H
2O
20.05ml become matrix liquid.The double antibody sandwich method running program:
0.1ml antibody HDL IgG bag is by (20 μ g/ml IgG spend the night for 4 ℃)
↓ washing
0.1ml sample (0.1ml urinates stoste) (37 ℃ 2 hours)
0.1ml lipoprotein titer HDL standard items (SigMA import) 0-2500ng/ml
↓ washing
0.1ml enzyme labelled antibody IgG (1: 2000) (37 ℃ 2 hours)
↓ washing
0.1ml matrix liquid (o-phenylenediamine 4mg is dissolved in 10ml)
Citric acid phosphoric acid hydrogen (37 ℃ 10 minutes)
The disodium damping fluid adds 3%H
2O
20.05ml)
↓
0.05ml 3M H
2SO
4Cessation reaction
↓
490nm surveys OD value (DG3022 enzyme-linked immunoassay instrument)
↓
Calculate the evaluation of the lipoprotein HDL of content (five) separation and purifying
It is single precipitation line that band lipoprotein HDL application agarose immunity double diffusion result centrifugal and purifying in genital areas demonstrates HDL, proves that antigen is purified, again through 0.22 μ degerming membrane filtration degerming.Measure protein content with the LowryShi method respectively and do antigen.Need the prepared fresh product when doing immunogene.(6) evaluation of IgG antibody
Through the HDL of twice purifying of saltouing IgG antibody, through laboratory diagnosis section of Changhai hospital serum chamber immunoelectrophoresis qualification result, HDL is single precipitation line.(7) preparation of typical curve
The HDL of doubling dilution (SigMA import) titer is made double antibody sandwich method ELISA and is measured, and draws canonical plotting respectively.Curve is " S " type, and sensitivity is good, and HDL-protein (HDL-P) lowest detection amount is about 100 μ g.Standard curve determination HDL-P content range is 100-1000ng/ml.All within normal person's content fluctuation range.The gradient concentration of the typical curve on the ELISA Plate of ELISA double antibody sandwich method is good.(8) precision test
For the precision of lipoprotein (HDL) ELISA of clear and definite and this research of proof, in having done batch, batch between and replica test between each hospital laboratory.
Test in batch and between criticizing, criticize interior mensuration as HDL respectively, the results are shown in Table 1 with pooled serum.Criticize an assay with pooled serum as HDL-P, the results are shown in Table 2.The gained coefficient of variation (CV%) illustrates that this law repeatability is good.
Table 2 ELISA method is measured in the criticizing of each lipoprotein and was tested classification extension rate n X (g/L) SD CV%LDL-P 1: 2,000 30 0.80 0.05 7.0
1∶4000 30 0.82 0.05 6.6HDL-P 1∶2000 30 0.27 0.03 5.0
1∶4000 30 0.23 0.02 10
Table 2 ELISA method is measured 1: 2,000 45 0.70 0.04 8.6HDL-P of experiment classification extension rate n X (g/L) SD CV%LDL-P accuracy experiment in 1: 2,000 30 0.22 0.02 9.0 (nine) between criticizing of each lipoprotein
ELISA method and UC method are surveyed the comparison of HDL-P (high-density lipoprotein (HDL)-protein).With 14 routine blood samples, every routine blood sample is used UC method and ELISA method separation determination HDL-P content respectively.
The UC method is with the 80P-7 of Hitachi type hydro-extractor and RPS50-2 rotor.Blood sampling 0.5ml is that the light gradient liquid of NaCL of 1.003-1.006 and density are that 1.35 the heavy gradient liquid of NaBr is made into linear gradient with density, 49000rpm, and 10～15 ℃ are centrifugal 4.5 hours.With constant flow pump four LP are with sucking-off (give dying with the acetyl Sudan black B earlier and be blue look).Measure HDL-P content respectively with the Lowry method.
The ELISA method is relevant with the highly significant as a result of a blood sample HDL-P content with UC method mensuration, and its related coefficient is r=0.96 (P＜0.01).The test of LP determination is urinated by Shanghai Changhai Hospital Nephrology dept. with kit of the present invention:
Increasing to the test item of range protein in the kidney patient urine at present, and the urine LP determination does not appear in the newspapers so far.This paper measures 55 routine various chronic kidney disease patients and urinates middle-high density lipoprotein (HDL) and low-density lipoprotein (LDL).And its clinical meaning tentatively inquired into.
Detected object is various chronic kidney disease patients, wherein nephrotic syndrome (ephrosis) 24 examples (I type 10 examples, II type 14 examples); Chronic renal failure (chronic kidney hypofunction) 19 examples, chronic nephritis 6 examples; Other ephrosis comprises latent nephritis, cirrhosis nephritis, lupus nephritis, gouty nephropathy and two kidney calcification, each 1 example of diabetic nephropathy.All detected objects all stay twenty-four-hour urine, measure urine middle-high density lipoprotein-protein (HDL-P) and low-density lipoprotein-protein (LDL-P) with enzyme linked immunosorbent assay (ELISA) method, are unit with μ g/L.Other establishes control group 19 examples, is normal adult.Result: one, the content of HDL-P and LDL-P in normal person's urine:
HDL-P and LDL-P are " 0 " in the control group normal person 19 example urine.Two, the content of HDL-P and LDL-P in nephropathy patient's urine:
Nephropathy patient's 24 examples, HDL-P total positives in the urine, wherein I type average out to 226 ± 178.09 μ g/L;
*Shanghai Medicine test center
The II type is 357.86 ± 172.14 μ g/L.I type urine LDL-P total negative (being 0); The II type is then all positive.Average 105.63 ± 112.96 μ g/L, and all in the case urine HDL-P all＞LDL-P, do not have 1 routine LDL-P＞HDL-P.Three, the content of HDL-P and LDL-P in chronic renal failure patients's urine:
Chronic renal failure patients's 19 examples, HDL-P positive person 16 examples (84%) in the urine, average out to 411.63 ± 392.24 μ g/L; LDL-P positive person 12 examples (63%) in the urine, average out to 353.33 ± 295.91 μ g/L.HDL-P in the urine＞LDL-P person's 5 examples (26%).Four, the content of chronic nephritis patient HDL-P and LDL-P:
Chronic nephritis patient's 6 examples, HDL-P and LDL-P are all positive in the urine, HDL-P average out to 3.01 ± 162.50 μ g/L, LDL-P average out to 164.50 ± 127.90 μ g/L.All in the cases urine HDL-P all＞LDL-P.Five, the content of HDL-P and LDL-P in other kidneys patient urine:
Other kidneys patient is totally 6 examples, and HDL-P is except that 1 routine lupus nephritis in the urine, and all the other 5 examples are all positive; But LDL-P has 3 examples negative (each 1 example of lupus nephritis, cirrhosis ephritis and gouty nephropathy), both equal positive persons, HDL-P＞LDL-P in the urine.Discuss:
Look in the check and see that HDL-P and LDL-P are all negative in the normal controls group urine, and the chronic kidney disease people be all the positive (both all positive or the two one of positive) person's 52 examples (95%), as seen urinate LP determination and can be used as one of index of chronic kidney damage.
In the ephrosis group of no obvious renal insufficiency, the positive rate that HDL-P detects in chronic nephritis and other kidneys patient urine is respectively 100%, 100% and 83%, and LDL-P then is 58%, 100% and 50%, the former average out to 94%, latter's average out to 69%.With regard to routine number, HDL-P positive person totally 35 examples (only 1 example is negative) in above-mentioned 3 groups of patients urine, and LDL-P positive person 23 examples (13 routine feminine gender) in the urine.LDL-P feminine gender in the 12 example urine is just arranged among the HDL-P positive person in urine.With regard to numerical value, in ephrosis group and the chronic nephritis group patient urine HDL-P mean value obviously＞LDL-P mean value (ephrosis group urine HDL-P mean value differs highly significant, P＜0.001 with urine LDL-P mean value); In each example urine of other ephrosis group HDL-P all＞LDL-P.So think for HDL-P in its urine of kidney trouble people detection of no obvious renal insufficiency than the LDL-P sensitivity.
Though in the chronic kidney hypofunction group urine HDL-P detect positive rate and mean value all＞(it is not remarkable that both mean values differ for LDL-P, P＞0.05), but wherein in the 5 example urine LDL-P obviously＞HDL-P (comprise urine HDL-P negative patient and urinate LDL-P460 μ g/L1 example), in 1 example in the urine HDL-P equate (being 500 μ g/L) with LDL-P.Learn by statistics to handle and prove with urine HDL-P or blood LDL-P all do not have correlativity.In addition both are all negative for 2 examples, but and blood urea nitrogen, serum creatinine or CrCl all do not have correlativity.
HDL-P was all positive during I type person urinated among the 24 routine nephropathy patients, and LDL-P is all negative; Both are all positive for II type person, so can be used as one of index of ephrosis I type and the discriminating of II type.C in lipoprotein inspection of I type urine and the urine
3Mensuration or disk electrophoresis detect albumin and match; Urine lipoprotein inspection of II type and hormone response match.The mechanism of this species diversity is like varying in size relevant with the garden shape particle diameter of two kinds of lipoprotein, HDL-P is 6.5～9.5mum, LDL is 20～25mum, HDL obviously＜LDL, it is generally acknowledged that I type glomerular filtration membrane damage is lighter, infer that it only allows the little HDL of particle diameter to pass through, and do not allow the big LDL of particle diameter to pass through; And II type glomerular filtration membrane damage is serious, and then the LDL that particle diameter is big also can pass through.So tentatively think the urine LP determination be can be used as one of selective proteinuria and nonselective proteinuria identification beacon.
Adopt the new bioengineering company limited in Shanghai to provide in ELISA method and the kit measurement urine thereof HDL-P and LDL-P a kind ofly simply fast, not need specific apparatus, wieldly be better than its method of inspection in the kidney trouble urine, and have the susceptibility and the specificity of height.So from methodology, the diagnosis and the somatotype of kidney trouble there is important clinical application value, should widely popularize.Xinhua Hospital Attached to Shanghai No.2 Medical Univ is measured the test of lipoprotein in the urine with the ELISA method:
In some disease, in the urine lipoprotein can appear.This paper is used for clinical detection 38 routine chronic kidney disease urine middle-high density lipoprotein (HDL) and low-density lipoproteins (LDL) with enzyme linked immunosorbent assay (being called for short ELISA) method first in my institute, now the result is announced as follows: one, method:
Medicine box with the new bioengineering company limited in Shanghai provides detects with the ELISA method.Two, object
(1) selects 3 age groups of normal person, 4～16 years old group (male 25 people, women 12 people), 17～23 years old group (male 28 people, women 17 people), 45～60 years old group (male 40 people, women 27 people).Measure wherein high-density lipoprotein (HDL)-protein (HDL-P) and low-density lipoprotein-protein (LDL-P) content.
(20 select normal person 15 people, chronic kidney disease 38 examples, wherein nephrotic syndrome 18 examples.Measure HDL-P and LDL-P content in its urine.Three, result
1.ELISA methodology is identified
(1) typical curve of HDL-P and LDL-P (shown in the typical curve that Fig. 1 ELISA method is surveyed lipoprotein), curve is " S " shape, and the scope of can surveying is between 80～1000ng/L, with the typical curve basically identical of Changhai hospital.
(2) my institute and Changhai hospital find that with being one group of blood sample result HDL-P content is significantly relevant (as shown in Figure 2) n=10, r=0.642, P＜0.05.It is relevant that LDL-P content (as shown in Figure 3) is highly significant.N＝10，r＝0.817，P＜0.01。
2. lipoprotein content in the normal human blood: HDL-P content in the blood, three age group content averages are more or less the same.LDL-P content average increased with the age.HDL-P and LDL-P content, all the women is higher than the male sex.
3. lipoprotein content during the normal person urinates: detect normal controls 15 examples, HDL-P and LDL-P are all negative in the urine.Detect nephrotic syndrome 18 examples, the results are shown in Table 3.HDL-P is positive in I type (8 example) the nephrotic syndrome urine, and LDL-P is negative, and HDL-P and LDL-P are all positive in II type (10 example) the nephrotic syndrome urine.Four, discuss
According to my academic test (examination) ELISA methods and results, be the typical curve and the Changhai hospital basically identical of " S " type.Be significantly relevant with making one group of sample.Illustrate that this method accuracy and repeatability are all better.
HDL-P and LDL-P content are consistent with the result of Changhai hospital in the normal human blood.
There is no HDL and LDL in the 15 routine normal persons urine, and nephrotic syndrome group patient, HDL only occurs in I type (light-duty) urine and HDL and LDL occur in II type (heavy type) urine.Because it is different to be spherical LP molecule grain size, the LDL molecular diameter is about 2～3 times of HDL, therefore the kind difference that LP occurs in the urine illustrates that not only the glomerulus Lu crosses film and suffer damage, but also the degree of its infringement can be described, so it is more more responsive than additive method with the ELISA method nephrotic syndrome to be carried out somatotype, and HDL is more responsive than LDL.The ELISA method is easy fast, and high specificity is highly sensitive, is used for clinical have bigger social benefit and economic benefit.HDL and LDL content (μ g/L) in the table 3 nephrotic syndrome urine
I type II type HDL-P LDL-P HDL-P LDL-P0 0 450 100,300 0 440 100,400 0 300 200,300 0 500 400,450 0 620 3,000 0 400 300,500 0 820 8,000 0 500 300
600 450
400 260 Shanghai Sixth Man people hospitals measure the test of lipoprotein in blood and the urine with the ELISA method:
The new method that detects lipoprotein component in nephropathy patient's urine with the ELISA method has been founded by the new bioengineering company limited in Shanghai.My institute is used for clinical with its medicine box that provides, feel more science of more original method, rationally.Now the result is announced as follows: one, method detects with the ELISA method with the medicine box that the new bioengineering company limited in Shanghai provides.Two, object
Select normal healthy people 10 people, acute and chronic kidney disease 28 examples, acute and chronic nephritis 8 examples wherein, nephrotic syndrome 10 examples, albuminuria 10 examples are measured HDL-P and LDL-P content in its urine.Three, result () ELISA methodology is identified
1.HDL-P reach the typical curve (as shown in Figure 4) of LDL-P, curve is " S " shape, the scope of can surveying is between 80～1000ng/L, with the typical curve basically identical of Changhai hospital.
2. my institute and Changhai hospital find that with being one group of blood sample result HDL-P content is significantly relevant (as shown in Figure 5), and LDL-P content is highly significant relevant (as shown in Figure 6).(2) detect healthy human urine's liquid 10 examples, HDL-P and LDL-P are all negative
Detect lipoprotein component content in the various ephrosis 28 routine urines, acute and chronic nephritis 3 examples wherein, the 3 routine HDL positives (positive rate 38%), the 2 routine LDL positives (positive rate 25%), nephrotic syndrome 10 examples, the 8 routine HDL positives, (positive rate 80%), the 5 routine LDL positives (positive rate 50%), albuminuria 10 examples, the 6 routine HDL positives (positive rate 60%), the 3 routine LDL positives (positive rate 30%) see attached list 4.
Lipoprotein content (μ g/L) acute and chronic nephritis nephrotic syndrome albuminuria HDL-P LDL-P HDL-P LDL-P HDL-P LDL-P--200---700-----360-640 100 in each kidney disease urine of table 4
*620 100300-640 250
*520---2,000 320
*450 200----300-400 320 450 80
*460-400 260 300-540 150
400 - - -
440 70
* - -
Annotate:
*The person is nephrotic syndrome II type four, discusses
According to my academic test (examination) ELISA method, be the typical curve and the Changhai hospital basically identical of " S " type.Be significantly relevant with making one group of sample.Illustrate that this method accuracy and repeatability are all better.There is no HDL and LDL among the 10 routine healthy human urines, HDL or (LDL) then appears in various kidney diseases in varying degrees.Meaningfully, in 10 routine nephrotic syndrome patients, only contain HDL all negative be that clinical diagnosis is the patient of I type (light-duty), this is because the impaired order of severity of glomerular filtration membrane differs and occur HDL and LDL in II type (heavy type) urine, and the HDL of small-molecular weight with the ELISA method nephrotic syndrome to be carried out somatotype for some reason more more responsive than additive method than easier leach former of LDL.When albuminuria and acute and chronic nephritis, we also can be according to the kind of LP in its urine and what, diagnose the order of severity of its state of an illness and prognosis or the like.Shanghai radio-immunity research institute of Tongji University measures the test of lipoprotein in people's urine with the ELISA method:
In some kidney trouble, dissimilar lipoprotein can occur in the urine, but not meet the report of this respect in the past.I at first use the new new bioengineering company limited in Shanghai to found usefulness enzyme linked immunosorbent assay (being called for short ELISA) method detection people urinate middle-high density lipoprotein (HDL) and low-density lipoprotein (LDL).
Now the result is announced as follows: one, method
Medicine box with the new bioengineering company limited in Shanghai provides detects with the ELISA method.Two, object
(1) selects 3 age groups of normal healthy people, 4～16 years old group (male 20 people, women 30 people), 17～23 years old group (male 25 people, woman 20 people) and organized (male 35 people, women 30 people) in 45～60 years old, measure wherein HDL2-protein (HDL-P) and low-density lipoprotein one protein (LDL-P) content.
(2) select normal healthy people 10 people, chronic kidney disease 27 examples, nephrotic syndrome 16 examples wherein, chronic nephritis 11 examples are measured HDL-P and LDL-P content in its urine.Three, result
1.ELISA methodology is identified
(1) typical curve of HDL-P and LDL-P (shown in the typical curve that Fig. 7 ELISA method is surveyed lipoprotein), curve is " S " shape.The scope of can surveying is between 80～1000ng/L, with the typical curve basically identical of Changhai hospital.
(2) I make one group of sample together with Changhai hospital at institute, found that HDL-P content is significantly relevant.N=10, r=0.726, P＜0.05 (as shown in Figure 8), it is relevant that LDL-P content is highly significant.N=10, r=0.866, p＜0.01 (as shown in Figure 9).
(3) HDL-P and LDL-P were " 0 " during lipoprotein component content was urinated in the detection normal healthy people urine.Detect nephrotic syndrome 16 examples, I type 7 examples wherein, II type 9 examples, lipoprotein content sees Table 5 in the urine.Detect chronic nephritis 11 examples, lipoprotein content sees Table 6 in the urine.
Lipoprotein content (μ g/L) in the table 1 nephrotic syndrome patient urine
I type II type HDL-P LDL-P HDL-P LDL-P450 0 300 200,300 0 500 400,400 0 100 400,440 0 100 240,300 0 220 210,450 0 300 1,600 0 530 110
700 230
800 250 4, discuss
Try out the ELISA methods and results according to me, be the typical curve and the Changhai hospital basically identical of " S " type.Be significantly relevant with making one group of sample.Illustrate that this method accuracy and repeatability are all good.There is no HDL and LDL among the 10 routine healthy human urines, and nephrotic syndrome 16 examples.
The content (μ g/L) of lipoprotein in the table 6 chronic nephritis patient urine
HDL-P LDL-P
400 320
0 0
400 260
500 250
0 0
0 0
100 0
0 0
250 100
0 0
600 320
The HDL positive has 15 examples in the urine, and positive rate is 93.7%, and the LDL positive has 9 examples, and positive rate is 60%, meaningfully, HDL only occurs in I type (light-duty) urine, and HDL and LDL occur in II type (heavy type) urine.Because it is different to be spherical LP molecule grain size, the LDL molecular diameter is about 2～3 times of HDL, therefore the kind difference that LP occurs in the urine has and only illustrates that the glomerulus Lu crosses film and suffer damage, but also the degree of its infringement can be described, so it is more more responsive than additive method with the ELISA method nephrotic syndrome to be carried out somatotype.Chronic nephritis patient's 11 examples, the 6 routine HDL positives wherein, positive rate is 54%, the 5 routine LDL positive, positive rate is 45%, can according to LP whether occurring in this patient's urine, and the kind that occurs, what of content are judged nephritis victim's state of an illness weight and prognosis.The ELISA method is easy fast, and high specificity is highly sensitive, is used for clinical have bigger social benefit and economic benefit.
Description of drawingsThe calibration curve Fig. 2 Xinhua Hospital Attached to Shanghai No.2 Medical Univ LPL-P correlation diagram Fig. 3 Xinhua Hospital Attached to Shanghai No.2 Medical Univ HDL-P correlation diagram Fig. 4 Shanghai Sixth Man people ELISA of hospital method of Fig. 1 Xinhua Hospital Attached to Shanghai No.2 Medical Univ ELISA method survey lipoprotein is surveyed the Shanghai radio-immunity HDL-P of research institute of calibration curve Fig. 8 Tongji University correlation diagram Fig. 9 Shanghai radio-immunity LPL-P of research institute of Tongji University correlation diagram of the Sixth Man people HDL-P of hospital correlation diagram Fig. 6 Shanghai, the calibration curve Fig. 5 Shanghai Sixth Man people LPL-P of hospital correlation diagram Fig. 7 Shanghai radio-immunity ELISA of research institute of Tongji University method survey lipoprotein of lipoprotein
EmbodimentOne, starting material and specification normal human serum animal thereof: new zealand white rabbit, healthy male, body weight 2～3kg ultracentrifuge: the board 80-P-7 of Hitachi type, RPZ-48T district band rotor, RPS-50-2 level
The rotor density gradient forms instrument, Hitachi's board, DGP-2 type Unic ultraviolet spectrometry degree meter: SP-800 type DG3022 enzyme-linked immunoassay instrument: Nanjing East China Electronics Co., Ltd pipe factory 721 spectrophotometer vortex mixers, XW-80 type PHS-20 type precision acidity meter electrophoresis apparatus (Shanghai) ELISA Plate, the ELISA of East China University of Science measures CV% (%)＜10% horseradish peroxidase (RZ3.0, SigMA) NaIO
4A.R (import packing) NaBH
4Other reagent Ns of A.R (import packing) a
2HPO
4.12H
2O A.R citric acid A.RNacl A.R glycerine chemical pure H
2O
2A.RTween-20 chemical pure o-phenylenediamine chemical pure H
2SO
4A.R distilled water must meet " the regulation High Density lipoprotein of Chinese pharmacopoeia (1990), HDL standard items (SigMA) two, coated antibody HDL Polyclonal Antibody Preparation () district band density gradient centrifugation purifying HDL operating process:
This paper is with density with reference to Hinton (1976) district, but has done bigger improvement.Use the 30-P-7 of Hitachi hydro-extractor, the RPZ-48T rotor is done band centrifugation.Use earlier Abbe refractometer, it is 1.4 liquid that NaBr gradient liquid is mixed with density, is that 1.4 NaBr and density are that 1.0 distilled water forms instrument through density gradient and added in district's band rotor by lateral opening then with density, and rotating speed maintains 2800/rpm at this moment.When adding to mesopore and having gradient liquid to flow out, to inject density with constant flow pump be 1.4 human serum 50ml through lateral opening again.After treating that blood sample all adds, rotating speed is risen to 42000/rpm obtain four peaks after centrifugal 18 hours altogether, receive sample by mesopore, sample flow records four lipoprotein component peaks through the Unic ultraviolet spectrophotometer.Wherein first peak is creamy white, and concentrates on first pipe, is very low density lipoprotein (VLDL) (vLDL).All the other several peaks all are light yellow, are respectively low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (HDL).All the other are degrease serum, contain other foreign protein peaks.With the peak of collecting, select the main peak few top pipe can not be too wide, in order to avoid sneak into intermediated-density lipoprotein (IDL).(2) preparation of lipoprotein antiserum HDL
Prepare protein Antiserum Preparation method routinely.At first the HDL with purifying is dissolved in the 0.01MPH7.4 phosphate buffer, add the Fu Shi Freund's complete adjuvant emulsification of equivalent after, give new zealand white rabbit, injection 2.0mg is fundamental immunity in nape portion multiple spot and the foot pad.Per two weeks are strengthened once, and immunity is 6 times altogether.The blood drawing test in 8-10 days of last immunity back is tired, and the satisfied person that tires collects antiserum by the heart blood drawing, and antiserum is again through ammonium sulfate (the 50% and 33% saturated (NH that saltouts for twice
4) SO
4) the fractional precipitation purifying becomes the anti-people of rabbit (HDL) IgG antibody (be called for short HDL IgG antibody).(3) preparation of the anti-people HDL of enzyme mark rabbit IgG antibody cross-linking agent.
The crosslinked acquisition of sodium periodate method " enzyme labelled antibody IgG " of improvement of the rabbit of purified gained anti-people HDL IgG antibody and horseradish peroxidase.Reagent preparation and operation steps are as follows: 1. sodium periodate labelling method reagent (1) 0.06M NaIO
4(0.13 gram NaIO
4, adding water to 10ml) and (2) 0.16M ethylene glycol solution 0.1ml adds water to 10ml (3) NaBH
4(5mg/ml, NaBH
45mg adds water to 1ml) (4) 0.05M PH9.5 carbonate buffer solution
First liquid: natrium carbonicum calcinatum 10.6 grams add water to 500ml
Second liquid: anhydrous sodium bicarbonate 16.8 grams add water to 1000ml
Get first liquid 16ml+ second liquid 34ml, add water to the 2. operation steps of periodic acid labelling method of 200ml (5) 0.02M PH7.4 PBS (with 0.1M PH7.4 PBS dilution)
7.5mg HRP+0.5ml dissolved in distilled water
↓
Add 0.06M NaIO
40.5ml, mix rearmounted 4 ℃, 30 minutes
↓
Add 0.16M ethylene glycol 0.5ml room temperature 30 minutes
↓
The rabbit lipotropism protein antibodies HDL IgG 0.5ml that adds mg/ml
(containing HDL IgG antibody 7mg approximately), mix back dress bag filter, use 0.05M, the pH9.6 carbonic acid buffer liquid of dialysing
↓
Sucking-off next day dialysate adds NaBH
4Solution 0.2ml (5mg/ml) put refrigerator 2 hours
↓
The above-mentioned bond mixed liquor of sucking-off adds the equal-volume saturated ammonium sulfate, behind 4 ℃ 30 ' in the refrigerator, abandons supernatant in centrifugal 4000 rev/mins * 15 minutes, and precipitation is dissolved in the liquid of dialysing among the 1ml PH7.4 0.02M PBS, changes liquid three times, each 1000ml
↓
Next day, the centrifugal again insolubles of removing promptly gets " enzyme labelled antibody IgG ", be sub-packed in the ampoule sealing after, put-40 ℃ of behaviour that preserve (four) lipoprotein HDL enzyme linked immunosorbent assay (ELISA) double antibody sandwich methods
Make the step urine sample: the diluted liquid of stoste bag: Na
2CO
30.16 gram, NaHCO
32.9 gram, NaN
30.02 gram adds water
Become the pH9.6 carbonate buffer solution to 100ml.Sample diluting liquid: NaCL 8 grams, KH
2PO
40.2 gram, Na
2HPO
42.9 gram, KCL0.2
Gram, T ween-20 0.5ml, NaN
30.2 gram adds water to 1000ml,
Add the 10ml calf serum with preceding every 100ml and become pH7.4PBS-Tween
20 sample diluting liquids.Cleansing solution: Tris 2.42g, 1N HCL 13ml, Tween-20 0.5ml adds water to 1000ml
Become pH7.4 0.02M Tris-Tween 20 cleansing solutions.Matrix liquid: 0.1M Na
2HPO
45.14ml (3.6 gram Na
2HPO
4Add water 100ml), 0.05M
Citric acid 4.86ml (1 gram citric acid adds water 100ml) o-phenylenediamine 4mg, 3%
H
2O
20.05ml become matrix liquid.The double antibody sandwich method running program:
0.1ml antibody HDL IgG bag is by (20 μ g/ml IgG spend the night for 4 ℃)
↓ washing
0.1ml sample (0.1ml urinates stoste) (37 ℃ 2 hours)
0.1ml lipoprotein titer HDL standard items (SigMA import) 0-2500ng/ml
↓ washing
0.1ml enzyme labelled antibody IgG (1: 2000) (37 ℃ 2 hours)
↓ washing
0.1ml matrix liquid (o-phenylenediamine 4mg is dissolved in 10ml)
Citric acid phosphoric acid hydrogen (37 ℃ 10 minutes)
The disodium damping fluid adds 3%H
2O
20.05ml)
↓
0.05ml 3M H
2SO
4Cessation reaction
↓
490nm surveys OD value (DG3022 enzyme-linked immunoassay instrument)
↓
Calculate the evaluation of the lipoprotein HDL of content (five) separation and purifying
It is single precipitation line that band lipoprotein HDL application agarose immunity double diffusion result centrifugal and purifying in genital areas demonstrates HDL, proves that antigen is purified, again through 0.22 μ degerming membrane filtration degerming.Measure protein content with the LowryShi method respectively and do antigen.Need the prepared fresh product when doing immunogene.(6) evaluation of IgG antibody
Through the HDL of twice purifying of saltouing IgG antibody, through laboratory diagnosis section of Changhai hospital serum chamber immunoelectrophoresis qualification result, HDL is single precipitation line.(7) preparation of typical curve
The HDL of doubling dilution (SigMA import) titer is made double antibody sandwich method ELISA and is measured, and draws canonical plotting respectively.Curve is " S " type, and sensitivity is good, and HDL-protein (HDL-P) lowest detection amount is about 100 μ g.Standard curve determination HDL-P content range is 100-1000ng/ml.All within normal person's content fluctuation range.The gradient concentration of the typical curve on the ELISA Plate of ELISA double antibody sandwich method is good.(8) precision test
For the precision of lipoprotein (HDL) ELISA of clear and definite and this research of proof, in having done batch, batch between and replica test between each hospital laboratory.
Test in batch and between criticizing, criticize interior mensuration as HDL respectively, the results are shown in Table 1 with pooled serum.Criticize an assay with pooled serum as HDL-P, the results are shown in Table 2.The gained coefficient of variation (CV%) illustrates that this law repeatability is good.
Table 1 ELISA method is measured in the criticizing of each lipoprotein and was tested classification extension rate n X (g/L) SD CV%LDL-P 1: 2,000 30 0.80 0.05 7.0
1∶4000 30 0.82 0.05 6.6HDL-P 1∶2000 30 0.27 0.03 5.0
1∶4000 30 0.23 0.02 10
Table 2 ELISA method is measured 1: 2,000 45 0.70 0.04 8.6HDL-P of experiment classification extension rate n X (g/L) SD CV%LDL-P accuracy experiment in 1: 2,000 30 0.22 0.02 9.0 (nine) between criticizing of each lipoprotein
ELISA method and UC method are surveyed the comparison of HDL-P (high-density lipoprotein (HDL)-protein).With 14 routine blood samples, every routine blood sample is used UC method and ELISA method separation determination HDL-P content respectively.
The UC method is with the 80P-7 of Hitachi type hydro-extractor and RPS50-2 rotor.Blood sampling 0.5ml is that the light gradient liquid of NaCL of 1.003-1.006 and density are that 1.35 the heavy gradient liquid of NaBr is made into linear gradient with density, 49000rpm, and 10～15 ℃ are centrifugal 4.5 hours.With constant flow pump four LP are with sucking-off (give dying with the acetyl Sudan black B earlier and be blue look).Measure HDL-P content respectively with the Lowry method.
The ELISA method is relevant with the highly significant as a result of a blood sample HDL-P content with UC method mensuration, and its related coefficient is r=0.96 (P＜0.01).
Claims (2)
1. a people urinates the content enzyme-linked immunosorbent assay (ELISA) reagent box of middle-high density lipoprotein, it is characterized in that this kit is made up of following reagent:
1. the HDL IgG antibody is wrapped in advance by 1 of plate
2. HDL IgG antibody enzyme labeling liquid is 1 bottle
3. HDL standard (2500,1250,625,56,10,0ng/ml) is each 1 bottle
4. concentrated cleaning solution is 1 bottle
5. substrate solution is towards each 1 bottle of liquid first, second
6. stop buffer is 1 bottle
2. preparation method that a kind of people as claimed in claim 1 urinates middle-high density lipoprotein content enzyme-linked immunosorbent assay (ELISA) reagent box, it is characterized in that this method comprises the following steps: one, starting material and specification normal human serum animal thereof: new zealand white rabbit, healthy male, body weight 2～3kg ultracentrifuge: the board 80-P-7 of Hitachi type, RPZ-48T district band rotor, the RPS-50-2 level
The rotor density gradient forms instrument, Hitachi's board, DGP-2 type Unic ultraviolet spectrometry degree meter: SP-800 type enzyme-linked immunoassay instrument: Labosystems Dragon Multiskam MK3UV754 spectrophotometer vortex mixer, XW-80 type PHS-20 type precision acidity meter electrophoresis apparatus, the Shanghai ELISA Plate, the ELISA of East China University of Science measures CV%＜10% horseradish peroxidase, RZ3.0, SigMANaIO
4A.R import packing NaBH
4Other reagent Ns of A.R import packing a
2HPO
412H
2O A.R citric acid A.RNacl A.R glycerine chemical pure H
2O
2A.RTween-20 chemical pure o-phenylenediamine chemical pure H
2SO
4A.R distilled water must meet " the regulation High Density lipoprotein of Chinese pharmacopoeia (1990), HDL standard items SigMA two, coated antibody HDL Polyclonal Antibody Preparation () district band density gradient centrifugation purifying HDL operating process:
This paper is with density with reference to Hinton 1976 districts, but has done bigger improvement.Use the 30-P-7 of Hitachi hydro-extractor, the RPZ-48T rotor is done band centrifugation, use earlier Abbe refractometer, it is 1.4 liquid that NaBr gradient liquid is mixed with density, be that 1.4 NaBr and density are that 1.0 distilled water forms instrument through density gradient and added in district's band rotor by lateral opening then with density, this moment, rotating speed maintained 2800/rpm, and when adding to mesopore and having gradient liquid to flow out, to inject density with constant flow pump be 1.4 human serum 50ml through lateral opening again.After treating that blood sample all adds, rotating speed is risen to 42000/rpm obtain four peaks after centrifugal 18 hours altogether, receive sample by mesopore, sample flow records four lipoprotein component peaks through the Unic ultraviolet spectrophotometer, wherein first peak is creamy white, concentrate on first pipe, be very low density lipoprotein (VLDL) vLDL, all the other several peaks all are light yellow, are respectively low-density lipoprotein LDL and high-density lipoprotein (HDL) HDL, and all the other are degrease serum, contain other foreign protein peaks, with the peak of collecting, select the main peak few top pipe can not be too wide, in order to avoid sneak into intermediated-density lipoprotein IDL; (2) preparation of lipoprotein antiserum HDL
Prepare protein Antiserum Preparation method routinely, at first the HDL with purifying is dissolved in the 0.01MPH7.4 phosphate buffer, add the Fu Shi Freund's complete adjuvant emulsification of equivalent after, give new zealand white rabbit, injection 2.0mg is fundamental immunity in nape portion multiple spot and the foot pad, and per two weeks are strengthened once, immunity is 6 times altogether, the blood drawing test in 8-10 days of last immunity back is tired, and the satisfied person that tires collects antiserum by the heart blood drawing, antiserum is saltoutd for twice through ammonium sulfate again, 50% and 33% saturated (NH
4) SO
4The fractional precipitation purifying becomes the anti-people HDL of rabbit IgG antibody, is called for short the HDL IgG antibody; (3) preparation of the anti-people HDL of enzyme mark rabbit IgG antibody cross-linking agent
The crosslinked acquisition of sodium periodate method " enzyme labelled antibody IgG " of improvement of the rabbit of purified gained anti-people HDL IgG antibody and horseradish peroxidase, reagent preparation and operation steps are as follows: I. sodium periodate labelling method reagent (1) 0.06M NaIO
4, 0.13 gram NaIO
4, add water to 10ml (2) 0.16M ethylene glycol solution 0.1ml and add water to 10ml (3) NaBH
4, 5mg/ml, NaBH
45mg adds water to 1ml (4) 0.05M PH9.5 carbonate buffer solution
First liquid: natrium carbonicum calcinatum 10.6 grams add water to 500ml
Second liquid: anhydrous sodium bicarbonate 16.8 grams add water to 1000ml
Get first liquid 16ml+ second liquid 34ml, add water to 200ml (5) 0.02M PH7.4 PBS, with the operation steps of 0.1M PH7.4 PBS dilution II. periodic acid labelling method
7.5mg HRP+0.5ml dissolved in distilled water
↓
Add 0.06M NaIO
40.5ml, mix rearmounted 4 ℃, 30 minutes
↓
Add 0.16M ethylene glycol 0.5ml room temperature 30 minutes
↓
The rabbit lipotropism protein antibodies HDL IgG 0.5ml that adds mg/ml
Contain HDL IgG antibody 7mg approximately, mix back dress bag filter, use 0.05M, the pH9.6 carbonic acid buffer liquid of dialysing
↓
Sucking-off next day dialysate adds NaBH
4Solution 0.2ml, 5mg/ml put refrigerator 2 hours
↓
The above-mentioned bond mixed liquor of sucking-off adds the equal-volume saturated ammonium sulfate, behind 4 ℃ 30 ' in the refrigerator, abandons supernatant in centrifugal 4000 rev/mins * 15 minutes, and precipitation is dissolved in the liquid of dialysing among the 1ml PH7.4 0.02M PBS, changes liquid three times, each 1000ml
↓
Next day, the centrifugal again insolubles of removing promptly gets " enzyme labelled antibody IgG ", be sub-packed in the ampoule sealing after, put-40 ℃ of behaviour that preserve (four) lipoprotein HDL enzyme linked immunosorbent assay ELISA double antibody sandwich methods
Make the step urine sample: the diluted liquid of stoste bag: Na
2CO
30.16 gram, NaHCO
32.9 gram, NaN
30.02 gram adds water
Become pH9.6 carbonate buffer solution sample diluting liquid: NaCL 8 to 100ml and restrain KH
2PO
40.2 gram, Na
2HPO
42.9 gram, KCL0.2
Gram, T ween-20 0.5ml, NaN
30.2 gram adds water to 1000ml,
Add the 10ml calf serum with preceding every 100ml and become pH7.4PBS-Tween
20 sample diluting liquid cleansing solutions: Tris 2.42g, 1N HCL 13ml, Tween-20 0.5ml adds water to 1000ml
Become pH7.4 0.02M Tris-Tween 20 cleansing solution matrix liquid: 0.1M Na
2HPO
45.14ml, 3.6 gram Na
2HPO
4Add water 100ml, 0.05M
Citric acid 4.86ml, 1 gram citric acid adds water 100ml o-phenylenediamine 4mg, 3%
H
2O
20.05ml become matrix liquid double antibody sandwich method running program:
0.1ml antibody HDLIgG wraps quilt, 20 μ g/ml IgG spend the night for 4 ℃
↓ washing
0.1ml sample 0.1ml urinates stoste, 37 ℃ 2 hours
0.1ml lipoprotein titer HDL standard items, SigMA import 0-2500ng/ml
↓ washing
0.1ml enzyme labelled antibody IgG 1: 2000,37 ℃ 2 hours
↓ washing
0.1ml matrix liquid, o-phenylenediamine 4mg is dissolved in 10ml
Citric acid phosphoric acid hydrogen, 37 ℃ 10 minutes
The disodium damping fluid adds 3%H
2O
20.05ml
↓
0.05ml 3M H
2SO
4Cessation reaction
↓
490nm surveys OD value, DG 3022 enzyme-linked immunoassay instrument
↓
Calculate the evaluation of the lipoprotein HDL of content (five) separation and purifying
It is single precipitation line that band lipoprotein HDL application agarose immunity double diffusion result centrifugal and purifying in genital areas demonstrates HDL, proves that antigen is purified, again through 0.22 μ degerming membrane filtration degerming.Measure protein content with the LowryShi method respectively and do antigen.Need the prepared fresh product when doing immunogene; (6) evaluation of IgG antibody
Through the HDL of twice purifying of saltouing IgG antibody, through laboratory diagnosis section of Changhai hospital serum chamber immunoelectrophoresis qualification result, HDL is single precipitation line; (7) preparation of typical curve
The HDL of doubling dilution, SigMA import titer is made double antibody sandwich method ELISA and is measured, draw canonical plotting respectively, curve is " S " type, and sensitivity is good, HDL-protein, HDL-P lowest detection amount is about 100 μ g, and standard curve determination HDL-P content range is 100-1000ng/ml.All within normal person's content fluctuation range, the gradient concentration of the typical curve on the ELISA Plate of ELISA double antibody sandwich method is good; (8) precision test
For the precision of the lipoprotein HDL ELISA of clear and definite and this research of proof, in having done batch, batch between and replica test between each hospital laboratory;
Test in batch and between criticizing, criticize interior mensuration as HDL respectively with pooled serum, the results are shown in Table 1, criticize an assay with pooled serum as HDL-P, the results are shown in Table 2, gained coefficient of variation CV% illustrates that this law repeatability is good;
Table 1 ELISA method is measured in the criticizing of each lipoprotein and was tested classification extension rate n X (g/L) SD CV%LDL-P 1: 2,000 30 0.80 0.05 7.0
1∶4000 30 0.82 0.05 6.6HDL-P 1∶2000 30 0.27 0.03 5.0
Table 2 ELISA method was measured 1: 2,000 45 0.70 0.04 8.6HDL-P of experiment classification extension rate n X (g/L) SD CV%LDL-P accuracy experiment in 1: 2,000 30 0.22 0.02 9.0 (nine) between criticizing of each lipoprotein in 1: 4,000 30 0.23 0.02 10
ELISA method and UC method are surveyed HDL-P, the comparison of high-density lipoprotein (HDL)-protein.With 14 routine blood samples, every routine blood sample is used UC method and ELISA method separation determination HDL-P content respectively;
The UC method is with the 80P-7 of Hitachi type hydro-extractor and RPS50-2 rotor.Blood sampling 0.5ml is that the light gradient liquid of NaCL of 1.003-1.006 and density are that 1.35 the heavy gradient liquid of NaBr is made into linear gradient with density, 49000rpm, and 10～15 ℃ are centrifugal 4.5 hours.With the sucking-off of four LP bands, elder generation gives dying with the acetyl Sudan black B and is blue look with constant flow pump.Measure HDL-P content respectively with the Lowry method;
The ELISA method is relevant with the highly significant as a result of a blood sample HDL-P content with UC method mensuration, and its related coefficient is r=0.96, P＜0.01.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01142683 CN1212517C (en) | 2001-12-18 | 2001-12-18 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying high density lipoprotein content in human urine and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01142683 CN1212517C (en) | 2001-12-18 | 2001-12-18 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying high density lipoprotein content in human urine and its preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1427257A true CN1427257A (en) | 2003-07-02 |
| CN1212517C CN1212517C (en) | 2005-07-27 |
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ID=4676912
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01142683 Expired - Fee Related CN1212517C (en) | 2001-12-18 | 2001-12-18 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying high density lipoprotein content in human urine and its preparation method |
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|---|---|
| CN (1) | CN1212517C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102368070A (en) * | 2011-07-01 | 2012-03-07 | 上海永昶医学诊断用品有限公司 | Kit for detecting content of MR-1S (Myofibrillogenesis Regulator-1S) in human plasma and preparation method thereof |
| CN102621306A (en) * | 2012-03-22 | 2012-08-01 | 中国人民解放军总医院 | Pepsin enzyme-linked immunoassay detection kit |
| CN103353533A (en) * | 2013-06-28 | 2013-10-16 | 武汉优尔生科技股份有限公司 | Human interleukin 35 enzyme-linked immunosorbent assay kit and preparation method thereof |
| CN103412130A (en) * | 2013-08-22 | 2013-11-27 | 四川省新成生物科技有限责任公司 | Reagent cup assembly, instrument and method for quickly determining content of specific proteins of urine |
| CN112029822A (en) * | 2020-08-21 | 2020-12-04 | 上海睿康生物科技有限公司 | Small and dense low-density lipoprotein cholesterol enzyme method detection kit |
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2001
- 2001-12-18 CN CN 01142683 patent/CN1212517C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102368070A (en) * | 2011-07-01 | 2012-03-07 | 上海永昶医学诊断用品有限公司 | Kit for detecting content of MR-1S (Myofibrillogenesis Regulator-1S) in human plasma and preparation method thereof |
| CN102621306A (en) * | 2012-03-22 | 2012-08-01 | 中国人民解放军总医院 | Pepsin enzyme-linked immunoassay detection kit |
| CN103353533A (en) * | 2013-06-28 | 2013-10-16 | 武汉优尔生科技股份有限公司 | Human interleukin 35 enzyme-linked immunosorbent assay kit and preparation method thereof |
| CN103353533B (en) * | 2013-06-28 | 2016-05-04 | 武汉云克隆科技股份有限公司 | HIL-3 5 enzyme-linked immunosorbent assay kits and preparation method thereof |
| CN103412130A (en) * | 2013-08-22 | 2013-11-27 | 四川省新成生物科技有限责任公司 | Reagent cup assembly, instrument and method for quickly determining content of specific proteins of urine |
| CN112029822A (en) * | 2020-08-21 | 2020-12-04 | 上海睿康生物科技有限公司 | Small and dense low-density lipoprotein cholesterol enzyme method detection kit |
| CN112485446A (en) * | 2020-11-18 | 2021-03-12 | 重庆中元汇吉生物技术有限公司 | Kit for measuring full-range C-reactive protein and preparation method thereof |
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|---|---|
| CN1212517C (en) | 2005-07-27 |
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