CN1212518C

CN1212518C - Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying low density lipoprotein content in human urine and its preparation method

Info

Publication number: CN1212518C
Application number: CN 01142684
Authority: CN
Inventors: 杜凤鸣
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-12-18
Filing date: 2001-12-18
Publication date: 2005-07-27
Anticipated expiration: 2021-12-18
Also published as: CN1427258A

Abstract

The present invention relates to the technical field of biotechnical products. The present invention discloses a kit for the enzyme linked immunosorbent assay on the content of low-density lipoprotein in human urine, and a preparation method. After being proved by the clinical test results, the kit of the present invention has accurate diagnosis on nephrotic patients, and the kit has the advantages of convenient and quick method, strong specificity, high sensitivity and good clinical application prospects.

Description

Low-density lipoprotein content enzyme-linked immunosorbent assay (ELISA) reagent box and preparation method in people's urine

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 (Lowdensity lipoprotein LDL) 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 LDL 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 LDL content enzyme and exempt from kit, this kit is with antibody LDL (IgG) bag quilt, and antibody LDL (IgG) connects horseradish peroxidase for detecting the sandwich method of antibody, detects the LDL content in people's urine.And form by following reagent:

1. low-density lipoprotein antibody IgG wraps in advance by 1 of plate

2. low-density lipoprotein antibody IgG enzyme labeling liquid is 1 bottle

3. the low-density lipoprotein antibody normal concentration is respectively 1 bottle of 2500ng/ml, 1250ng/ml, 625ng/ml, 56ng/ml, 10ng/ml, 0ng/ml

4. wrap diluted liquid: Na ₂CO ₃0.16 gram, NaHCO ₃0.29 gram, NaN ₃0.02 gram, adding water to 100ml becomes the pH9.6 carbonate buffer solution

5. sample diluting liquid: NaCL 8 restrains 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, adds PBS-Tween 20 sample diluting liquids that the 10ml calf serum becomes pH7.4 with preceding every 100ml

6. cleansing solution: Tris 2.42g, 1N HCL 13ml, Tween-20 0.5ml add water to 0.02M Tris-Tween 20 cleansing solutions that 1000ml becomes pH7.4

7. matrix liquid: 0.1M Na ₂HPO ₄5.14ml, 0.05M citric acid 4.86ml, o-phenylenediamine 4mg, 3%H2O20.05ml become matrix liquid

8. stop buffer: 3M H ₂SO ₄

Another technical matters to be solved of the present invention has provided the preparation method of the ELISA kit of LDL content in the above-mentioned urine, and this method comprises the following steps:

One, starting material and specification thereof

Normal human serum

Animal: 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 horizontal 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 MK3

The UV754 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 ₄A.R (import packing)

Other reagent:

Na ₂HPO ₄.12H ₂O A.R

Citric acid A.R

Nacl A.R

The glycerine chemical pure

H ₂O ₂ A.R

The Tween-20 chemical pure

The o-phenylenediamine chemical pure

H ₂SO ₄ A.R

Distilled water must meet " the regulation of Chinese pharmacopoeia (1990)

Low Density lipoprotein, low-density lipoprotein standard items (SigMA)

Two, coated antibody low-density lipoprotein Polyclonal Antibody Preparation

(1) district's band density gradient centrifugation purifying low-density lipoprotein 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, NaBr gradient liquid is mixed with the liquid that density is 1.4mg/ml, be that the NaBr of 1.4mg/ml and distilled water that density is 1.0mg/ml form instrument through density gradient and added in district's band rotor by lateral opening then with density, this moment, rotating speed maintained 2800rpm.When adding to mesopore and having gradient liquid to flow out, to inject density with constant flow pump be the human serum 50ml of 1.mg/ml4 through lateral opening again.After treating that blood sample all adds, rotating speed is risen to 42000rpm 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) (v low-density lipoprotein).All the other several peaks all are light yellow, are respectively low-density lipoprotein (low-density lipoprotein) and high-density lipoprotein (HDL) (HDL).All the other are degrease serum, contain other foreign protein peaks;

(2) preparation of lipoprotein antiserum low-density lipoprotein

Prepare protein Antiserum Preparation method routinely.At first the low-density lipoprotein with purifying is dissolved in the 0.01M PH7.4 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 becomes the anti-people of rabbit (low-density lipoprotein) IgG antibody (being called for short low-density lipoprotein antibody IgG) through the ammonium sulfate fractional precipitation purifying of saltouing for twice again.

(3) preparation of the anti-human ldl IgG antibody of enzyme mark rabbit cross-linking agent

The crosslinked acquisition of sodium periodate method " enzyme labelled antibody IgG " of improvement of anti-human ldl IgG antibody of the rabbit of purified gained 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, add water to 10ml)

(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 200ml

(5) 0.02M PH7.4 PBS (with 0.1M PH7.4 PBS dilution)

2. the operation steps of 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 IgG 0.5ml that adds mg/ml

(containing low-density lipoprotein antibody IgG 7mg approximately), mix back dress bag filter, use 0.05M, pH9.5

The 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, and 4 ℃ in refrigerator was abandoned supernatant in centrifugal 4000 rev/mins * 15 minutes after 30 seconds, 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 preservations

(4) behaviour of lipoprotein low-density lipoprotein enzyme linked immunosorbent assay (ELISA) double antibody sandwich method

Make step

Urine sample: stoste

Wrap diluted liquid: Na ₂CO ₃0.16 gram, NaHCO ₃0.29 gram, NaN ₃0.02 gram, adding water to 100ml becomes the pH9.5 carbonate buffer solution.

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, adding the 10ml calf serum with preceding every 100ml becomes pH7.4PBS-Tween 20 sample diluting liquids.

Cleansing solution: Tris 2.42 grams, 1M HCl 13ml, Tween-20 0.5ml add water to 1000ml becomes 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 the IgG antibody 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 low-density lipoprotein 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 content

(5) evaluation of the lipoprotein low-density lipoprotein of separation and purifying

It is single precipitation line that band lipoprotein low-density lipoprotein application agarose immunity double diffusion result centrifugal and purifying in genital areas demonstrates low-density lipoprotein, proves that antigen is purified, again through 0.22 μ m 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 low-density lipoprotein antibody IgG of twice purifying of saltouing, through laboratory diagnosis section of Changhai hospital serum chamber immunoelectrophoresis qualification result, low-density lipoprotein is single precipitation line.

(7) preparation of typical curve

The low-density lipoprotein 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 (low-density lipoprotein-P) the lowest detection amount is 100 μ g to low-density lipoprotein-protein.Standard curve determination low-density lipoprotein-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 (low-density lipoprotein) 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 low-density lipoprotein respectively, the results are shown in Table 1 with pooled serum.Make low-density lipoprotein-P with pooled serum and criticize an assay, 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 batch interior experiment of each lipoprotein

Classification extension rate n X (g/L) SD CV%

Low-density lipoprotein-P 1: 2,000 30 0.80 0.05 7.0

1∶4000 30 0.82 0.05 6.6

HDL-P 1∶2000 30 0.27 0.03 5.0

1∶4000 30 0.23 0.02 10

Table 2 ELISA method is measured experiment between criticizing of each lipoprotein

Classification extension rate n X (g/L) SD CV%

Low-density lipoprotein-P 1: 2,000 45 0.70 0.04 8.6

HDL-P 1∶2000 30 0.22 0.02 9.0

(9) accuracy experiment

ELISA method and UC method are surveyed the comparison of low-density lipoprotein-P (low-density lipoprotein-protein).

With 14 routine blood samples, every routine blood sample is used UC method and ELISA method separation determination low-density lipoprotein-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 (being blue look with the acetyl sudan black B stain).Measure low-density lipoprotein-P content respectively with the Lowry method.

Highly significant is relevant as a result with a blood sample with UC method mensuration for the ELISA method, and its related coefficient is r=0.975 (P＜0.01).

The test of LP determination is urinated by Shanghai Changhai Hospital Nephrology dept.:

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 (low-density lipoprotein).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 (low-density lipoprotein-P), be unit with μ g/L with enzyme linked immunosorbent assay (ELISA) method.Other establishes control group 19 examples, is normal adult.The result:

One, the content of HDL-P and low-density lipoprotein-P in normal person's urine:

HDL-P and low-density lipoprotein-P are " 0 " in the control group normal person 19 example urine.

Two, the content of HDL-P and low-density lipoprotein-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;

The II type is 357.86+172.14 μ g/L.I type urine low-density lipoprotein-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＞low-density lipoprotein-P, do not have 1 routine low-density lipoprotein-P＞HDL-P.

Three, the content of HDL-P and low-density lipoprotein-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; Low-density lipoprotein-P positive person 12 examples (63%) in the urine, average out to 353.33 ± 295.91 μ g/L.HDL-P＞low-density lipoprotein in the urine-P person's 5 examples (26%).

Four, the content of chronic nephritis patient HDL-P and low-density lipoprotein-P:

Chronic nephritis patient's 6 examples, HDL-P and low-density lipoprotein-P are all positive in the urine, HDL-P average out to 3.01 ± 162.50 μ g/L, low-density lipoprotein-P average out to 164.50 ± 127.90 μ g/L.All in the cases urine HDL-P all＞low-density lipoprotein-P.

Five, the content of HDL-P and low-density lipoprotein-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 low-density lipoprotein-P has 3 examples negative (each 1 example of lupus nephritis, cirrhosis ephritis and gouty nephropathy), both equal positive persons, HDL-P＞low-density lipoprotein-P in the urine.

Discuss:

Look in the check and see that HDL-P and low-density lipoprotein-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.

Ephrosis group at 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%, low-density lipoprotein-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 low-density lipoprotein-P positive person 23 examples (13 routine feminine gender) in the urine.Low-density lipoprotein-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＞low-density lipoprotein-P mean value (ephrosis group urine HDL-P mean value differs highly significant, P＜0.001 with urine low-density lipoprotein-P mean value); In each example urine of other ephrosis group HDL-P all＞low-density lipoprotein-P.So think for HDL-P in its urine of kidney trouble people detection of no obvious renal insufficiency than low-density lipoprotein-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 low-density lipoprotein-P, P＞0.05), but wherein in the 5 example urine low-density lipoprotein-P obviously＞HDL-P (comprise urine HDL-P negative patient and urinate low-density lipoprotein-P460 μ g/L1 example), in 1 example in the urine HDL-P equate (being 500 μ g/L) with low-density lipoprotein-P.Learn by statistics to handle and prove with urine HDL-P or blood low-density lipoprotein-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 low-density lipoprotein-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, low-density lipoprotein is 20～25mum, HDL obviously＜low-density lipoprotein, 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 low-density lipoprotein of particle diameter to pass through; And II type glomerular filtration membrane damage is serious, and then the low-density lipoprotein 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 low-density lipoprotein-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 (low-density lipoprotein) 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 the low-density lipoprotein-protein (content of low-density lipoprotein-P).

(2) select normal person 15 people, chronic kidney disease 38 examples, wherein nephrotic syndrome 18 examples.Measure HDL-P and low-density lipoprotein-P content in its urine.

Three, result

1.ELISA methodology is identified

(1) typical curve of HDL-P and low-density lipoprotein-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 low-density lipoprotein-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.Low-density lipoprotein-P content average increased with the age.HDL-P and low-density lipoprotein-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 low-density lipoprotein-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 low-density lipoprotein-P is negative, and HDL-P and low-density lipoprotein-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 low-density lipoprotein-P content are consistent with the result of Changhai hospital in the normal human blood.

There is no HDL and low-density lipoprotein in the 15 routine normal persons urine, and nephrotic syndrome group patient, HDL only occurs in I type (light-duty) urine and HDL and low-density lipoprotein occur in II type (heavy type) urine.Because it is different to be spherical LP molecule grain size, the low-density lipoprotein 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 low-density lipoprotein.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 low-density lipoprotein content (μ g/L) in the table 3 nephrotic syndrome urine

The I type The II type

HDL-P low-density lipoprotein-P HDL-P low-density lipoprotein-P

0 0 450 100

300 0 440 100

400 0 300 200

300 0 500 400

450 0 620 300

0 0 400 300

500 0 820 800

0 0 500 300

600 450

400 260

Shanghai Sixth Man people hospital measures 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

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 low-density lipoprotein-P content in its urine.

Three, result

(1) the ELISA methodology is identified

1.HDL-P reach the typical curve (as shown in Figure 4) of low-density lipoprotein-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 low-density lipoprotein-P content is highly significant relevant (as shown in Figure 6).

(2) detect healthy human urine's liquid 10 examples, HDL-P and low-density lipoprotein-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 low-density lipoprotein positives (positive rate 25%), nephrotic syndrome 10 examples, the 8 routine HDL positives, (positive rate 80%), the 5 routine low-density lipoprotein positives (positive rate 50%), albuminuria 10 examples, the 6 routine HDL positives (positive rate 60%), the 3 routine low-density lipoprotein positives (positive rate 30%) see attached list 4.

Lipoprotein content (μ g/L) in each kidney disease urine of table 4

Acute and chronic nephritis Nephrotic syndrome Albuminuria

HDL-P low-density lipoprotein-P HDL-P low-density lipoprotein-P

HDL-P low-density lipoprotein-P

- - 200 - - -

700 - - - - -

360 - 640 100 ^* 620 100

300 - 640 250 ^* 520 -

- - 2000 320 ^* 450 200

- - - - 300 -

400 320 450 80 ^* 460 -

400 260 300 - 540 150

400 - - -

440 70 ^* - -

Annotate: ^*The person is a nephrotic syndrome II type

Four, discuss

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 low-density lipoprotein among the 10 routine healthy human urines, HDL or (low-density lipoprotein) then appear 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 low-density lipoprotein 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 low-density lipoprotein.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 (low-density lipoprotein).

Now the result is announced as follows:

One, method

Two, object

(1) selects 3 age groups of normal healthy people, 4～16 years old group (male 20 people, woman 30 people), 17～23 years old group (male 25 people, woman 20 people) and organized (male 35 people in 45～60 years old, woman 30 people), measure wherein HDL2-protein (HDL-P) and the low-density lipoprotein-protein (content of low-density lipoprotein-P).

(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 low-density lipoprotein-P content in its urine.

Three, result

1.ELISA methodology is identified

(1) typical curve of HDL-P and low-density lipoprotein-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 low-density lipoprotein-P content is highly significant.N=10, r=0.866, p＜0.01 (as shown in Figure 9).

(3) HDL-P and low-density lipoprotein-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 5 nephrotic syndrome patient urine

The I type The II type

HDL-P low-density lipoprotein-P HDL-P low-density lipoprotein-P

450 0 300 200

300 0 500 400

400 0 100 400

440 0 100 240

300 0 220 210

450 0 300 160

0 0 530 110

700 230

800 250

Four, 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 low-density lipoprotein 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 low-density lipoprotein-P

400 320

0 0

400 260

500 250

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 low-density lipoprotein positive has 9 examples, and positive rate is 60%, meaningfully, HDL only occurs in I type (light-duty) urine, and HDL and low-density lipoprotein occur in II type (heavy type) urine.Because it is different to be spherical LP molecule grain size, the low-density lipoprotein 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 low-density lipoprotein positive, positive rate is 45%, LP whether occurs in can urinating according to this patient, 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 drawings

Fig. 1 Xinhua Hospital Attached to Shanghai No.2 Medical Univ ELISA method is surveyed the typical curve of lipoprotein

Fig. 2 Xinhua Hospital Attached to Shanghai No.2 Medical Univ low-density lipoprotein-P correlogram

Fig. 3 Xinhua Hospital Attached to Shanghai No.2 Medical Univ HDL-P correlogram

Fig. 4 Shanghai Sixth Man people ELISA of hospital method is surveyed the typical curve of lipoprotein

Fig. 5 Shanghai Sixth Man people HDL-P of hospital correlogram

Fig. 6 Shanghai Sixth Man people hospital low-density lipoprotein-P correlogram

The Shanghai radio-immunity ELISA of research institute of Fig. 7 Tongji University method is surveyed the typical curve of lipoprotein

The Shanghai radio-immunity HDL-P of research institute of Fig. 8 Tongji University correlogram

Shanghai radio-immunity research institute of Fig. 9 Tongji University low-density lipoprotein-P correlogram

Embodiment

One, starting material and specification thereof

Normal human serum

Animal: new zealand white rabbit, healthy male, body weight 2～3kg

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 spectrophotometers

Vortex mixer: XW-80 type

PHS-20 type precision acidity meter

Electrophoresis apparatus: Shanghai

Horseradish peroxidase (RZ3.0, SigMA)

NaIO ₄A.R (import packing)

NaBH ₄A.R (import packing)

Other reagent:

Na ₂HPO ₄.12H ₂O A.R

Citric acid A.R

Nacl A.R

The glycerine chemical pure

H ₂O ₂ A.R

The Tween-20 chemical pure

The o-phenylenediamine chemical pure

H ₂SO ₄ A.R

Distilled water must meet " the regulation of Chinese pharmacopoeia (1990)

Low Density lipoprotein, low-density lipoprotein standard items (SigMA)

Two, coated antibody low-density lipoprotein Polyclonal Antibody Preparation

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) (v low-density lipoprotein).All the other several peaks all are light yellow, are respectively low-density lipoprotein (low-density lipoprotein) 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 low-density lipoprotein

Prepare protein Antiserum Preparation method routinely.At first the low-density lipoprotein with purifying is dissolved in the 0.01M PH7.4 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 (low-density lipoprotein) IgG antibody (be called for short low-density lipoprotein antibody IgG).

The crosslinked acquisition of sodium periodate method " enzyme labelled antibody IgG " of improvement of anti-human ldl IgG antibody of the rabbit of purified gained and horseradish peroxidase.Reagent preparation and operation steps

As follows:

1. 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 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 200ml

(5) 0.02M PH7.4 PBS (with 0.1M PH7.4 PBS dilution).

2. the operation steps of 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 IgG 0.5ml that adds mg/ml

(containing low-density lipoprotein antibody IgG 7mg approximately), mix back dress bag filter, use 0.05M, pH9.6

The carbonic acid buffer liquid of dialysing

↓

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 preservations

Make step

Urine sample: stoste

Wrap diluted liquid: Na ₂CO ₃0.16 gram, NaHCO ₃0.29 gram, NaN ₃0.02 gram, adding water to 100ml becomes the pH9.6 carbonate buffer solution

Sample diluting liquid: NaCL 8 grams, KH ₂PO ₄0.2 gram, Na ₂HPO ₄2.9 gram, KCL 0.2 gram, T ween-20 0.5ml, NaN ₃0.2 gram adds water to 1000ml, adding the 10ml calf serum with preceding every 100ml becomes pH7.4PBS-Tween 20 sample diluting liquids

Cleansing solution: Tris 2.42g, 1N HCL 13ml, Tween-20 0.5ml add water to 1000ml becomes 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.

Table I double antibody sandwich method running program

0.1ml the IgG antibody bag is by (20 μ g/ml IgG spend the night for 4 ℃)

↓ washing

0.1ml sample (0.1ml urinates stoste) (37 ℃ 2 hours)

↓ 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 content

It is single precipitation line that band lipoprotein low-density lipoprotein application agarose immunity double diffusion result centrifugal and purifying in genital areas demonstrates low-density lipoprotein, 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

(7) preparation of typical curve

(7) precision test

Test in batch and between criticizing, criticize interior mensuration as low-density lipoprotein respectively, the results are shown in Table 1 with pooled serum.Make low-density lipoprotein-P with pooled serum and criticize an assay, the results are shown in Table 3.The gained coefficient of variation (CV%) illustrates that this law repeatability is good.

Table 1 ELISA method is measured batch interior experiment of each lipoprotein

Classification extension rate n X (g/L) SD CV%

Low-density lipoprotein-P 1: 2,000 30 0.80 0.05 7.0

1∶4000 30 0.82 0.05 6.6

HDL-P 1∶2000 30 0.27 0.03 5.0

1∶4000 30 0.23 0.02 10

Classification extension rate n X (g/L) SD CV%

Low-density lipoprotein-P 1: 2,000 45 0.70 0.04 8.6

HDL-P 1∶2000 30 0.22 0.02 9.0

(9) accuracy experiment

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 low-density lipoprotein-P content respectively with the Lowry method.

Claims

1. the content enzyme-linked immunosorbent assay (ELISA) reagent box of low-density lipoprotein in the people urine is characterized in that this kit is made up of following reagent:

1. low-density lipoprotein antibody IgG wraps in advance by 1 of plate

2. low-density lipoprotein antibody IgG enzyme labeling liquid is 1 bottle

5. sample diluting liquid: NaCL 8 restrains KH ₂PO ₄0.2 gram, Na ₂HPO ₄2.9 gram, KCL 0.2 gram, Tween-20 0.5ml, NaN ₃0.2 gram adds water to 1000ml, adds PBS-Tween 20 sample diluting liquids that the 10ml calf serum becomes pH7.4 with preceding every 100ml

7. matrix liquid: 0.1M Na ₂HPO ₄5.14ml, 0.05M citric acid 4.86ml, o-phenylenediamine 4mg, 3%H ₂O ₂0.05ml become matrix liquid

8. stop buffer: 3M H ₂SO ₄