CN102221619A - ELISA (Enzyme-Linked Immuno Sorbent Assay) detection method and kit of cryptosporidium virus capsid protein antibody - Google Patents

Abstract

The invention provides an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection method and a kit of a cryptosporidium virus capsid protein antibody, and is used for detecting the cryptosporidium infection serum antibodies of animals and human beings, wherein cryptosporidium virus capsid proteins are used as an antigen to be coated on an ELISA plate; and the standard positive serum and standard negative serum of the cryptosporidium virus capsid proteins, a substrate color developing solution, a cleaning solution, a stop solution, an ELISA substance diluting solution, a sample diluting solution and a confining solution form the ELISA kit. The invention has the beneficial effect that the detected sample is serum, has the advantages of high detection specificity, high sensitivity and good stability, can detect the infection of the cryptosporidium of the human beings and is suitable for the rapid diagnosis in clinic and the large-scale application of the epidemiological survey in grassroots, villages and the like.

Description

Cryptosporidium viral capsid proteins antibody ELISA detection method and kit

Technical field

The invention provides a kind of Cryptosporidium viral capsid proteins antibody ELISA detection method and kit, be used for the detection of animal and people's Cryptosporidium spp serum antibody, the invention also discloses the preparation method of above-mentioned ELISA kit, belong to serology detection technique field.

Background technology

Cryptosporidium is ubiquitous intestines and stomach parasitic protozoa, can cause diarrhoea human and domestic animal.Contain the ight soil of the mankind, domestic animal, pet and the wild animal of Cryptosporidium egg capsule, directly threatening human drinking water safety, cause Cryptosporidiosis (Cryptosporidiosis).It is to be caused by people Cryptosporidium and Cryptosporidum parvum (Cryptosporidium parvum, C.parvum genotype II) that the molecule epidemic disease-ology research result demonstrates most of people's Cryptosporidiosis.At present, the Cryptosporidium spp that does not also have effective method treatment people.And direct microscopy of existing diagnostic method and dyeing microscopic examination method are time-consuming, effort, recall rate are low; IFA and PCR method are had relatively high expectations to instrument and equipment and reagent, therefore set up a kind of fast, specificity is higher and directly the diagnostic method of result of determination be very necessary.

Cryptosporidium virus (Cryptosporidium parvum virus, Cryspovirus, CSpV) be to find in the C.parvum egg capsule in calf, separated of Khramtsov in 1997, this virus contains long dsRNA (L-dsRNA) and short dsRNA (S-dsRNA), wherein S-dsRNA coding viral capsid proteins.Afterwards, Khramtsov finds that again all C.hominis and C.parvum contain this dsRNA virus, and does not exist in other kinds of Cryptosporidium.Therefore can be the characterization of molecules of CSpV and antigenic characteristic as a foundation that detects C.hominis and C.parvum egg capsule.And this viral capsid proteins gene S-dsRNA just can be used as molecular labeling that the people is had that infective Cryptosporidium (C.hominis and C.parvum) and other kind Cryptosporidium differentiate and the foundation of separating and identifying that contains viral worm strain.

Still do not have at present and utilize anti-Cryptosporidium virus to detect the report of antibody in the infection animal serum.

Summary of the invention

The invention discloses antibody ELISA detection kit in a kind of Cryptosporidium viral capsid proteins serum, solved the shortcoming that current ight soil detection sensitivity is low, time-consuming, require great effort, can detect the Cryptosporidium of infected person.

The present invention also provides the preparation method of above-mentioned ELISA detection kit, utilizes the S-dsRNA albumen of expressing at Escherichia coli E.coli BL21 (DE3) as antigen, has set up the indirect ELISA method of antibody in the Cryptosporidium spp serum that detects the people.

Antibody ELISA detection method in the Cryptosporidium viral capsid proteins serum of the present invention is characterized in that: antigen is Cryptosporidium virus capsid recombinant protein, is used for detecting serum Cryptosporidium virus antibody.

Cryptosporidium viral capsid proteins antibody indirect ELISA detection kit of the present invention, composition comprises: Cryptosporidium virus standard positive serum, Cryptosporidium virus standard female serum, ELIAS secondary antibody, bag are by the ELISA Plate of viral capsid proteins, substrate colour developing liquid, cleansing solution, stop buffer, confining liquid and enzyme mark thing dilution and sample diluting liquid.

The preparation method of last kit, it is characterized in that: with the Cryptosporidium viral capsid proteins is antigen coated in ELISA Plate, Cryptosporidium virus protein standard positive serum and standard female serum, substrate colour developing liquid, cleansing solution, stop buffer, enzyme mark thing dilution, sample diluting liquid and confining liquid are formed the ELISA kit.

Described antigen is Cryptosporidium virus capsid recombinant protein.

ELISA kit test method of the present invention may further comprise the steps:

(1) sample pre-treatments: tested serum is diluted with sample diluting liquid at 1: 200;

(2) detect with ELISA kit of the present invention, in wrapping, add confining liquid by the ELISA Plate hole of Cryptosporidium virus capsid recombinant protein, add serum again, hatched 60 minutes, the cleansing solution washing, add ELIAS secondary antibody, hatched 60 minutes, the cleansing solution washing adds colour developing liquid and hatched 10 minutes, the stop buffer cessation reaction is measured absorbance with microplate reader;

(3) analyzing and testing result:

When OD490nm ≧ 0.32, sample is judged to be the positive; When OD490nm ﹤ 0.268, sample is judged to be feminine gender.

Good effect of the present invention is: the sample of detection is a serum, and detection specificity height, highly sensitive, good stability can detect the infection of people Cryptosporidium, are suitable for clinical quick diagnosis and epidemiology survey large-scale application such as basic unit, rural area.

Description of drawings

Fig. 1 is the purifying SDS-PAGE electrophoretogram of recombinant protein;

Among the figure: M: the low molecular weight protein (LMWP) mark; 1: the recombinant protein of purifying; M:DL 2000 Marker; Lane1:the protein purfied.

Embodiment

The following example is intended to further illustrate, rather than restriction the present invention.It will be appreciated by those skilled in the art that, under the prerequisite that does not deviate from the spirit and principles in the present invention, all will fall in the claim scope that awaits the reply of the present invention any parallel change of the present invention and change.

Embodiment 1

Cryptosporidium viral capsid proteins CSpV expresses

1 material and method

1.1 bacterial strain Cryptosporidium virus CSpV capsid protein is expressed bacterium pET-28a (+)-S.

Expression, purifying and the renaturation of Cryptosporidium virus CSpV capsid protein

The ratio of CSpV capsid protein positive expression bacterium in 1 % is inoculated in the 500 mL LB nutrient culture media, and 37 ℃ of shaken cultivation 2 ~ 3 h are to bacterium liquid OD ₆₀₀The nm value is 0.4 ~ 0.6, and adding IPTG is 1 mmol/L to final concentration, and 4 h are cultivated in continuation, after abduction delivering finishes, collect bacterium liquid.Carry out the purifying of destination protein according to the Ni-NTA instructions.With containing 8,6,4,2,0 mol/L urea, the PBS solution of 0.5 mol/L L-Arg carries out gradient dialysis renaturation.Measure protein concentration with BCA protein content assay kit.

1.3 the preparation of mouse-anti Cryptosporidium positive serum

Get 2 * 10 of purifying ⁷Individual Cryptosporidum parvum egg capsule is dissolved among the PBS, Ultrasonic Pulverization, during at interval sampling, till microscopically be cannot see egg capsule, centrifugal 20 min of 12 000 r/min, supernatant is antigen.Measure protein concentration with BCA protein content assay kit.With the BALB/c mouse (100 μ g/ only) in age 66 ~ 8 weeks of this antigen peritoneal immunity, immune programme for children prepares positive serum routinely.

The result

2.1 the purification of recombinant proteins positive expression bacterium of Cryptosporidium virus CSpV capsid protein is behind the IPTG abduction delivering, with Ni-NTA affinity column purifying destination protein.The destination protein of purifying is analyzed through SDS-PAGE, as seen at 37 ku places a single protein band is arranged, and sees accompanying drawing 1.

Embodiment 2

With the recombinant protein is the indirect ELISA method that antigen is set up antibody in the Cryptosporidium spp serum that detects the people

1 material and method

1.1, the selection of sealer

Use the recombinant protein coated elisa plate, 4 ℃ are spent the night.With 10 % hyclones, 10 % normal rabbit serums, 5 % skimmed milk powers is sealer, and with the PBST dilution, every hole adds 200 μ L, hatches 2 h for 37 ℃.Washing rear enclosed 2 h add positive serum contrast and negative serum contrast respectively, hatch 1 h for 37 ℃.Washing, the sheep anti mouse two that adds the HRP mark is anti-, hatches 1 h for 37 ℃.The washing back adds substrate, observes change color.After the colour developing, with 2 mol/L H ₂SO ₄Cessation reaction.Microplate reader is measured OD ₄₉₀The nm value.Establish positive control (P), negative control (N) and blank simultaneously, P/N ﹥ 2 is judged to the positive.Select the sealing effect the best.

1.2 determining of an antigen and an anti-working concentration

Recombinant protein coated elisa plate with variable concentrations 1,0.5,0.25,0.125,0.0125,0.00 625 μ g/ hole, positive serum with different extension rate 1:200,1:400,1:800,1:1 600,1:3 200 is hatched, carry out indirect ELISA, determine antigen and an anti-best effort concentration.

1.3 determining of the sheep anti mouse two anti-working concentrations of HRP mark

Select best sealer, an antigen and an anti-best effort concentration dilution of pressing are variable concentrations 1:500,1:1 000,1:2 000,1:4 000,1:8 000,1:16 000 with the two anti-dilutions of HRP mark sheep anti mouse.Add the substrate buffer solution colour developing, measure OD ₄₉₀The nm value is determined two anti-best effort concentration.

1.4 determining of indirect ELISA criterion as a result

Detect 12 parts of healthy mice serum, measure OD ₄₉₀The nm value, calculating mean value

With standard deviation SD.

The result

2.1 the foundation of Cryptosporidum parvum antibody test ELISA method

According to the result who measures, selecting best confining liquid is the PBST solution of 5 % skimmed milk powers, best antigen coated concentration 0.25 μ g/ hole, tested serum dilution 1: 200, and sheep anti mouse two anti-extension rate 1:2 000 are optimal detection condition.

2.2 determining of indirect ELISA criterion as a result

Detect 12 parts of healthy mice serum OD through indirect ELISA ₄₉₀Nm value mean value

Be 0.216, standard deviation SD is 0.026, so positive detects down and is limited to

+ 3SD=0.294.In order to reduce false positive and false-positive appearance, critical value is added and subtracted a standard deviation as suspicious interval.Therefore work as OD ₄₉₀Nm ≧ 0.32 o'clock, sample is judged to be the positive; Work as OD ₄₉₀During nm ﹤ 0.268, sample is judged to be feminine gender; As 0.268 ≦ OD ₄₉₀During nm ﹤ 0.32, sample is judged to be the positive, but needs to detect again.If when heavily examining, OD ₄₉₀Nm ≧ 0.294, sample is judged to be the positive; OD ₄₉₀Nm ﹤ 0.294, sample are judged to be feminine gender (seeing attached list 1).

Determining of subordinate list 1 indirect ELISA criterion as a result

Embodiment 3

Antibody indirect ELISA kit assembling in detection people's the Cryptosporidium spp serum

1, Cryptosporidium virus standard positive serum, Cryptosporidium virus standard female serum

2, bag is by viral recombinant protein ELISA Plate

3, the sheep anti mouse two of HRP mark is anti-

4. substrate colour developing liquid: A liquid: the pH value is that 0.1mol/L sodium acetate-citrate buffer solution of 5.0 is mixed with 0.3% hydrogen peroxide storing solution.B liquid: tetramethyl benzidine, be prepared into 0.2mg/mL solution earlier, be that sodium acetate-citrate buffer solution of 5.0 is mixed with 0.2mg/L solution with the 0.1mol/LpH value.

5. cleansing solution: the pH value is the PBST damping fluid of 7.4 0.1% Tween-20.

6. enzyme mark thing dilution: the pH value is the PBST damping fluid of 7.4 0.01% Tween-20.

7. sample diluting liquid: the pH value is the PBST damping fluid of 7.4 0.1% Tween-20.

8. stop buffer: 2mol/L sulfuric acid.

Test example 1

The indirect ELISA method performance measurement

1 material and method

1.1 material

The mouse-anti cryptosporidium andersoni ( C.andersoni) positive serum, the arc worm of mouse-anti ( T.gondii) positive serum, mouse-anti Lan Shi giardia lamblia ( G.lamblia) positive serum, the tender eimeria tenella of mouse-anti chicken ( E.tenella) all preparations according to a conventional method of positive serum.

1.2 method

1.2.1 sensitivity tests is with mouse-anti C.parvumPositive serum is pressed 1:200,1:400,1:800,1:1 600,1:3 200 doubling dilutions, uses the indirect ELISA of having set up, determines its susceptibility.

1.2.2 the specificity test is with mouse-anti C.parvumPositive serum and negative serum are contrast, measure mouse-anti with the indirect ELISA method of having set up C.andersoniPositive serum, mouse-anti T.gondiiPositive serum mouse, anti- G.lambliaPositive serum, mouse-anti E.tenellaPositive serum is observed serum cross reaction situation, determines the specificity of reaction.

1.2.3 replica test detects 7 parts of positive serum samples in same block of plate, every part of blood serum sample repeats 3 holes, measures according to the running program of indirect ELISA, calculates every part of blood serum sample OD ₄₉₀The coefficient of variation of nm value (CV) is with the repeatability of detected sample in the inspection lot; Other get 3 with the purification of recombinant proteins bag by good ELISA Plate, detect 7 parts of positive serum samples under the same conditions, measure according to the running program of indirect ELISA, calculate with a blood serum sample OD between different plates ₄₉₀The coefficient of variation of nm value (CV) is with the repeatability of detected sample between inspection lot.

The susceptibility that subordinate list 2 ELISA detect

2. result

2.1 sensitivity tests can be drawn by subordinate list 2, when positive serum is diluted to 1: 1 600 the time, OD ₄₉₀Nm ≧ 0.32, sample still decidable are positive, when positive serum is diluted to 1:3 200, and OD ₄₉₀Nm ﹤ 0.268, sample is judged to be feminine gender.Therefore the susceptibility of indirect ELISA detection method is 1:1 600, shows that the susceptibility of this method is higher.

3.2 specificity test

Detect mouse-anti with the indirect ELISA method of setting up C.andersoniPositive serum, mouse-anti T.gondiiPositive serum mouse, anti- G.lambliaPositive serum, mouse-anti E.tenellaPositive serum is all negative, and no cross reaction shows this method specificity strong (seeing attached list 3).

3.3 revision test coefficient of variation average was 2.59 % in replica test was criticized, revision test coefficient of variation maximal value is 10.5% between batch, shows that this method has repeatability (seeing attached list 4).

The specificity that table 3 ELISA detects

Repeated experiment result in the group of table 4 indirect ELISA, between group

Test example 2

Actual sample detects

1 material and method

1.1 8 healthy BALB/c mouse are raised in the detection of serum to be checked test, behind the immunosupress 5d, irritate stomach inoculation 10 respectively at the 6th d ⁵Individual purifying C.parvumEgg capsule.Tail vein blood behind the inoculation 20d detects with above-mentioned indirect ELISA method.Other gets 20 healthy BALB/c mouse and above-mentioned mouse is together raised 20 d, and tail vein blood detects with above-mentioned indirect ELISA method then.

The result

The detection of serum to be checked is tested with above-mentioned indirect ELISA method 8 parts of infection C.parvumThe mice serum of egg capsule detects, and the result shows that 8 parts of blood serum samples are all positive; In other 20 parts of mice serums, 13 parts of positive serums, 7 parts of negative serums, positive rate are 65%.

Claims (5)

1. antibody ELISA detection method in the Cryptosporidium viral capsid proteins serum, it is characterized in that: antigen is Cryptosporidium virus capsid recombinant protein, can detect Cryptosporidium virus antibody in the serum.

2. Cryptosporidium viral capsid proteins antibody indirect ELISA detection kit, composition comprises: Cryptosporidium virus standard positive serum, Cryptosporidium virus standard female serum, ELIAS secondary antibody, bag are by the ELISA Plate of viral capsid proteins, substrate colour developing liquid, cleansing solution, stop buffer, confining liquid and enzyme mark thing dilution and sample diluting liquid.

Such as claim 2 the preparation method of kit, it is characterized in that: with the Cryptosporidium viral capsid proteins is antigen coated in ELISA Plate, Cryptosporidium virus protein standard positive serum and standard female serum, substrate colour developing liquid, cleansing solution, stop buffer, enzyme mark thing dilution, sample diluting liquid and confining liquid are formed the ELISA kit.

4. the preparation method of the described ELISA kit of claim 3 is characterized in that: described antigen is Cryptosporidium virus capsid recombinant protein.

5. ELISA kit test method may further comprise the steps:

(1) sample pre-treatments: tested serum is diluted with sample diluting liquid at 1: 200;

(2) detect with the described ELISA kit of claim 2, in wrapping, add confining liquid by the ELISA Plate hole of Cryptosporidium virus capsid recombinant protein, add serum again, hatched 60 minutes, the cleansing solution washing, add ELIAS secondary antibody, hatched 60 minutes, the cleansing solution washing adds colour developing liquid and hatched 10 minutes, the stop buffer cessation reaction is measured absorbance with microplate reader;

(3) analyzing and testing result:

When OD490nm ≧ 0.32, sample is judged to be the positive; When OD490nm ﹤ 0.268, sample is judged to be feminine gender.