CN102680681A - Novel I-type human immunodeficiency virus (HIV-1) infection enzyme test-free reagent kit - Google Patents

Abstract

The invention provides a novel I-type human immunodeficiency virus (HIV-1) infection enzyme test-free reagent kit. The reagent kit comprises A, HIV-1gp41 protein, B, a blank enzyme linked plate, C, acid eluent, D, goat anti-human immunoglobulin G (IgG) marked with horseradish peroxidase, E, tetramethyl benzidine (TMB) color development solutions and F, stop solutions.

Description

A kind of I type human immunodeficiency virus (HIV-1) New Development infects the inspection-free test agent box of enzyme

Technical field:

The present invention relates to a kind of immunity detection reagent; Its composition, preparation and application thereof; Be particularly related to a kind of I type human immunodeficiency virus (HIV-1) New Development and infect inspection-free test agent box of enzyme (the affine force method of restricted antigen) and preparation method thereof, this kit is mainly used in the recent infection of on population level, distinguishing HIV-1 and infects with long-term.

Background technology:

Prospective cohort study in the epidemiological method be considered to detect " goldstandard " that the HIV New Development infects, but owing to need great manpower, time and input, cohort study can't widespread use always.In addition, cohort study exists to lose to be visited, and therefore semi-open property or open formation are because there is bigger variation in the monitoring crowd, in case uncontrollable good formation quality, whole formation skew will occur even lose representativeness.What is more important; In formation observation process, the behavior that the crowd that follows up a case by regular visits to often receives is in various degree easily disturbed, thereby underestimates target group's HIV New Development infection rate; And this influence tends to along with the carrying out of cohort study constantly deepen accumulation; Thereby cause HIV New Development infection rate downward trend gradually to occur, infect the accurate judgement of people for epidemic situation, measure employs prevention best opportunity of control measure.

Just be based on the above-mentioned defective of cohort study, be the basis with the cross-section survey sample, detecting the method for estimating HIV New Development infection rate through the laboratory, its advantage is able to highlight.Its cardinal rule is: human body is after infected by HIV; The mark relevant with the infection incident can appear; Used and detected whether these marks exist or the variation of quality/quantity, and " window phase " of employed method of detecting infection recently, can judge it is the length of HIV-1 infection time.The window phase that newly advances the monitoring of infection method is different from the window phase that in the past said HIV detects, and it is meant from patient infection HIV and begins, and the infected is judged to be a period of time (averaging usually) of infecting recently; The HIV detection window phase is meant that then patient infection HIV is early stage, and all detection methods fail it is judged to be positive a period of time (blind area).Except that the HIV nucleic acid detection method; All these methods all can be referred to as STARHS (serological testing algorithm for recent HIV seroconversion; HIV infects serological detection method recently), propose in 1998 by Janssen etc. the earliest.Because in cross-sectional study, the crowd does not receive investigation and disturbs, as long as reasonably define the target group, the HIV New Development infection rate of being estimated can well be represented this crowd's true popularity.Another huge advantage of this method is that the pharmacy people have kept historical sample, just can obtain the historical data of HIV New Development infection rate.Change and judge infection time length owing to the STARHS method is based on the various indexs that occur behind the organism infection HIV; But human body is because various factors; Particularly antiviral therapy, disease process, individual congenital/influences such as Strain hypotype of acquired immunity situation and infection; Can cause the variation of These parameters to be interfered or occur repeatedly; Thereby can not keep absolute linear relationship with infection time, therefore all STARHS methods all can not be used for diagnosis of case at present, promptly can not return the result to individuality; And only limit to the investigation and the scientific research of public health angle, mainly be behavioural characteristic and the hazards analysis etc. of detection, the New Development infection population of HIV New Development infection rate.

The HIV that occurs in recent years method of detecting infection recently all is to the HIV-1 type, mainly contains following several kinds:

1, the BED-CEIA:BED-CEIA full name is that HIV-1BED catches the EIA method, is called for short the BED method

2, band immunoblot experiment

3, IgG3 antibody

4, agglutination test

5, the infection recently based on saliva sample detects

6, affinity test

At present; Have only U.S. Sedia company can produce like product; The HIV-1 New Development that the said firm produces infects detection kit (BED method) and has been widely used in HIV-1 New Development infection detection range, but in use there is the false determination ratio problem of higher in this method.According to correlative study, I type human immunodeficiency virus (HIV-1) New Development infects the inspection-free test agent box of enzyme (the affine force method of restricted antigen) and can address the above problem preferably.

Summary of the invention:

On the basis based on restricted antigen affinity principle, a kind of low false determination ratio, steady quality are provided, satisfy the preparation method that the HIV-1 New Development infects the kit that detects.

Behind the human infection HIV-1, the affinity of anti-HIV-1 specific antibody can increase along with the prolongation of infection time in the serum.The present invention has adopted the restricted antigen I gG trapped enzyme of single hole linked immunosorbent assay, measures the affinity of specific antibody.Concrete principle is as follows:

1. at first use Recombinant HIV-1 specific antigen (gp41) that to discern multiple hypotype to encapsulate elisa plate; The adding blood serum sample is placed on 37 ° of C and hatches 60min; At this moment, all HIV-1 specific antibody (antibody that comprises low-affinity and high-affinity) gp41 albumen that all can be coated on the elisa plate is caught.

2. add 37 ° of C reactions of acid eluent 15min, the antibody of low-affinity (IgG) will come off from elisa plate.

3. the goat anti-human igg who adds horseradish peroxidase-labeled behind 37 ° of C reaction 30min, forms the anti-human IgG compound of gp41-HIV-1 specific IgG (high-affinity)-HRP mark.

4. add 25 ° of C reactions of TMB colour developing liquid 15min.Recent infection presents different colored intensities with the long-term sample that infects.

5. measure the OD value in every hole, use the ODn of the calibration object calculation sample in the kit then, coming judgement sample according to the value of ODn is recent infection or long-term the infection.

For this reason, the present invention provides a kind of I type human immunodeficiency virus (HIV-1) New Development to infect the inspection-free test agent box of enzyme, and kit of the present invention comprises following component:

HIV-1gp41 albumen;

Blank elisa plate;

Acid eluent;

The goat anti-human igg of horseradish peroxidase-labeled;

The TMB liquid that develops the color;

Stop buffer.

Wherein,

HIV-1gp41 albumen is the gp41 chimeric protein, can buy from market to obtain.As: available from the luxuriant and rich with fragrance roc in Shenzhen.

Blank elisa plate is the blank elisa plate of NUNC, can buy from market to obtain.As: available from Thermo company.

Acid eluent is: 0.01M citrate buffer (pH=3.0).

Enzyme conjugates is the enzyme conjugates dilution that contains the goat anti-human igg; The PBS damping fluid (pH=7.2) that wherein said enzyme conjugates dilution is 0.05M wherein contains 1% bovine serum albumin(BSA), 1% sucrose; 0.1% Tween-20,0.1% Triton-100 and 0.1% Proclin-300.

TMB colour developing liquid is the tetramethyl biphenyl amine aqueous solution, can buy from market to obtain, as: available from KPL company.

Stop buffer is the sulfuric acid solution of 1N.

Kit of the present invention is that said components is prepared, and is mixed with the separately unit of packing that is fit to use, and the unit that comprises of the present invention is following:

①	Encapsulate elisa plate
		②	20 * lavation buffer solution
③	Sample diluting liquid
		④	Acid eluent
⑤	Enzyme conjugates
		⑥	Colour developing liquid
⑦	Stop buffer
		⑧	Strong positive contrast (HPC)
⑨	Weak positive control (LPC)
		⑦	Calibration object (CAL)
⑧	Negative control (NC)

The preparation method who wherein encapsulates elisa plate is following:

Preparation technology

Encapsulate: the pH that uses 0.05M is that 6.0 phosphate buffer is diluted to 0.063 μ g/mL with gp41 albumen, and the damping fluid after will diluting then joins in the microwell plate of 96T, and every hole adds 100 μ L, places 4 ° of C to encapsulate 18～22h microwell plate then;

Wash plate: use robotization to wash plate machine washing plate 2 times, drain;

Sealing: every hole adds 200 μ L confining liquids, then microwell plate is placed 4 ° of C sealing 18～22h; Dry: use robotization to wash the plate machine microwell plate is drained, dried overnight, drying condition is 18～25 ° of C of temperature, humidity < 30%.

Pack: the elisa plate that will encapsulate is put into aluminium foil bag, encapsulates after putting into a drying agent, places under 4 ° of C conditions and preserves.

The prescription that encapsulates the various liquid that use in the process is following:

Coating buffer prescription: the PBS damping fluid (pH=6.0) of 0.05M;

The confining liquid prescription: the PBS damping fluid (pH=6.0) of 0.05M, wherein contain 0.5% casein, 2% sucrose, 1% glycocoll, 0.1% Proclin-300;

The PBS damping fluid (pH=7.2) of lotion prescription: 0.05M wherein contains 0.05% Tween-20; Pure water is all adopted in all liq preparation.

Wherein

The preparation method of 20 * lavation buffer solution is following: the PBS damping fluid (pH=7.2) of 0.1M, wherein contain 0.1% Tween-20;

The preparation method of sample diluting liquid is following: the PBS damping fluid (pH=6.0) of 0.05M, wherein contain 0.5% casein, 2% sucrose, 1% glycocoll and 0.1% Proclin-300.

Acid eluent is 0.01M citrate buffer (pH=3.0).

Enzyme conjugates is for containing the enzyme conjugates dilution of certain density goat anti-human igg antibody (available from KPL company); Wherein said enzyme conjugates is the PBS damping fluid (pH=7.2) of 0.05M; Contain 1% bovine serum albumin(BSA), 1% sucrose, 0.1% Tween-20 and 0.1% Triton-100.

Developer: available from KPL company.

The sulfuric acid solution of stop buffer: 1N.

The strong positive contrast is: OD value scope is the HIV-1 type positive serum of 0.600-2.100

Weak positive control is: OD value scope is the HIV-1 type positive serum of 0.200-0.800

Calibration object is: OD value scope is the HIV-1 type positive serum of 0.300-0.900

Negative control is: OD value scope is the HIV-1 type positive serum of 0.000-0.250

Kit of the present invention, the loading amount of each component can confirm according to applicable cases, so that loading amount is appropriately for well, as is mixed with 96 person-portions, 192 person-portions, 480 person-portions etc.

Can be according to following method packing like 192 person-portions:

Various component loading amounts require as follows:

The component title	Component concentration
		20 * lavation buffer solution	60mL
Sample diluting liquid	60mL
		Acid eluent	60mL
Enzyme conjugates	25mL
		Colour developing liquid	25mL
Stop buffer	25mL
		Strong positive contrast (HPC)	?50μL
Weak positive control (LPC)	0μL
		Calibration object (CAL)	50μL

Negative control (NC)

?50μL

Assembling:

Each unit decals is known, be assembled into complete kit, be stored under the 2-8 ℃ of condition with-20 ° of C.

The method of application of kit of the present invention is following:

The sample that the present invention is suitable for is the serum or the blood plasma of liquid.Institute's specimen is the positive sample of HIV-1.

1. test design

During detection, use two hole negative controls, three hole calibration objects, reference substance.Primary dcreening operation test, each sample are carried out single hole and are detected;

3 hole duplicate detection are carried out in validation test, each sample; Add master drawing shown in table 1 and table 2.

The application of sample order of table 1 primary dcreening operation test

NC	HPC	6	14	22	30	38	46	54	62	70	78
												NC	HPC	7	15	23	31	39	47	55	63	71	79
CAL	HPC	8	16	24	32	40	48	56	64	72	80
												CAL	1	9	17	25	33	41	49	57	65	73	81
CAL	2	10	18	26	34	42	50	58	66	74	82
												LPC	3	11	19	27	35	43	51	59	67	75	83
LPC	4	12	20	28	36	44	52	60	68	76	84
												LPC	5	13	21	29	37	45	53	61	69	77	85

Table 2 validation test application of sample order

NC	HPC	2	5	8	10	13	16	18	21	24	26
												NC	HPC	3	5	8	11	13	16	19	21	24	27
CAL	HPC	3	6	8	11	14	16	19	22	24	27
												CAL	1	3	6	9	11	14	17	19	22	25	27
CAL	1	4	6	9	12	14	17	20	22	25	28
												LPC	1	4	7	9	12	15	17	20	23	25	28
LPC	2	4	7	10	12	15	18	20	23	26	28
												LPC	2	5	7	10	13	15	18	21	23	26	Blank

2. concrete test procedure

A. 500 μ L sample diluting liquids are added the diluted sample pipe, add 5 μ L (1:101) reference substances then, calibration object and sample to be checked use pipettor to blow and beat (4～6 times) repeatedly, and be fully subsequent use behind the mixing.Negative control is prepared 2 parts, and strong positive contrast, calibration object and weak positive control are respectively prepared 3 parts;

B. according to shown in the above-mentioned application of sample table, every hole adds good each the 100 μ L of reference substance, calibration object and sample to be checked of dilution, covers the shrouding film, and 37 ℃ (± 1 ℃) are hatched 60min.The single hole check is all carried out in primary dcreening operation test, every duplicate samples, and the application of sample order is as shown in table 1.The reinspection of three holes is carried out in validation test, every duplicate samples, and the application of sample order is as shown in table 2.

C. discard liquid in each hole, use 96 holes to wash plate machine washing plate 4 times, add washing lotion 400 μ L/ holes at every turn, wash plate at every turn and left standstill 10 seconds, clap and do;

D. every hole adds the acid eluent of 200 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 15min

E. repeating step C;

F. every hole adds the enzyme conjugates of 100 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 30min;

G. repeating step C;

H. every hole adds 100 μ L colour developing liquid, puts 25 ℃ of (± 1 ℃) incubations 15 minutes;

J. every hole adds 100 μ L stop buffers, selects ELIASA wavelength 450nm, and reference wavelength 630nm measures each hole OD value.

[explanation of test findings]

1. primary dcreening operation test: if the ODn of sample >=2.0, this sample is long-term infection sample, if sample ODn 2.0, then need carry out validation test (concrete operations see the validation test part for details);

2. validation test: < 2.0 sample need carry out validation test to primary dcreening operation test ODn.In the validation test, each sample carries out three holes to be rechecked, and validation test application of sample order is as shown in table 2, and concrete test procedure sees [test method] for details.If<1.0, this sample is the recent infection sample to the ODn of validation test sample, if sample ODn>=1.0 think that then this sample is the long-term sample that infects;

3. the result explains: the early-stage Study according to the scientist of U.S. Disease Control and Prevention Center shows that the CUTOFF value is that ODn=1.0 representes that the average positive commentaries on classics time is 141 days.But can distinguish to some extent in different regions, China still needs and will more test and data analysis the average positive commentaries on classics time.

[calculating of New Development infection rate]

By U.S. disease prevention and control center and global AIDS coordinating office, UNAIDS and World Health Organization's whole world AIDS and venereal disease working group combine recommendation, the computing formula of New Development infection rate that is used for the transversal section sample is following:

$I_F=\frac{R-(\mathrm{FRR}*P)}{(1-\mathrm{FRR})\mathrm{\&omega;N}}\&times;100$

I _FRepresent the New Development infection rate;

Negative sample quantity in the N representative investigation;

Positive sample size in the P representative investigation;

R represents the quantity that is judged as the sample that infects recently in the positive sample;

ω represents average window phase;

FRR represents false recent infection rate.

Can find out that from computing formula window phase ω and false recent infection rate FRR influence the whether accurate key influence factor of New Development infection rate estimation.Therefore, when using this formula calculating New Development infection rate, recommend following three preconditions:

A) there is suitable sample crowd to be used to estimate New Development infection rate and local FRR coefficient, obtain the FRR value of this area or own country, and this correction coefficient accurately and reliably;

B) there is the infected's of antiviral therapy history sample to foreclose, do not include the calculating of New Development infection rate in;

C) do not meet the sample of including testing conditions in, as far as possible deletion.

Description of drawings:

Fig. 1 is testing process figure

Embodiment

Below further specify the present invention through embodiment, but not as limitation of the present invention.

Embodiment 1

[main composition]

[condition of storage]

This kit has refrigeration package and frozen-pack.

The condition of storage of refrigeration package: 2～8 ℃

The condition of storage of frozen-pack :≤-20 ° of C

[being suitable for instrument]

1. automatic ELIAS microplate washer, ELIASA

2. pipettor

3. oscillator

4. diluted sample pipe

5. timer

6. incubator (37 ° of C and 25 ° of C)

[sample requirement]

1. the suitable sample of this product is the serum or the blood plasma of liquid.

2. this product test sample is the positive sample of HIV-1.

3. sample was no more than for 2 weeks 2～8 ° of C following storage times of condition.Standing storage must be at-20 ° of C or following.

4. avoid sample repeatedly, repeatedly freeze thawing, can not use the sample of significant hemolysis and microbial contamination.

5. before using, can through put upside down (6～8 times) repeatedly or with oscillator with the abundant mixing of sample.

[exclusion standard]

For obtaining testing result more accurately, can not include detection in from following crowd's sample:

1. AIDS patient;

2. the person that accepts the antiviral therapy;

3.CD4 cell is less than 200;

4. Diagnostic Time was greater than the infected of 6 months.

[preparation before the test]

1. with all kits, calibration object and reference substance place room temperature (18～30 ° of C) 60min at least.TMB places 25 ° of C incubators.Immediately kit is put back under the condition of storage after the use.

2. prepare 1 * washing lotion

20 * washing lotion of 60ml is fully mixed (can use magnetic stirrer 15min) with the deionized water of 1140ml.

Attention:, must melt back (can use water-bath to carry out the heating of short time) fully in crystallization and dilute if occur crystallization in 20 * washing lotion.1 * washing lotion can be used a week at ambient temperature.

[test method]

2. test design

During detection, use two hole negative controls, three hole calibration objects, reference substance.Primary dcreening operation test, each sample are carried out single hole and are detected;

3 hole duplicate detection are carried out in validation test, each sample; Add master drawing shown in table 1 and table 2.

The application of sample order of table 1 primary dcreening operation test

NC	HPC	6	14	22	30	38	46	54	62	70	78
												NC	HPC	7	15	23	31	39	47	55	63	71	79
CAL	HPC	8	16	24	32	40	48	56	64	72	80
												CAL	1	9	17	25	33	41	49	57	65	73	81
CAL	2	10	18	26	34	42	50	58	66	74	82
												LPC	3	11	19	27	35	43	51	59	67	75	83
LPC	4	12	20	28	36	44	52	60	68	76	84
												LPC	5	13	21	29	37	45	53	61	69	77	85

Table 2 validation test application of sample order

NC	HPC	2	5	8	10	13	16	18	21	24	26
												NC	HPC	3	5	8	11	13	16	19	21	24	27
CAL	HPC	3	6	8	11	14	16	19	22	24	27
												CAL	1	3	6	9	11	14	17	19	22	25	27
CAL	1	4	6	9	12	14	17	20	22	25	28
												LPC	1	4	7	9	12	15	17	20	23	25	28
LPC	2	4	7	10	12	15	18	20	23	26	28
												LPC	2	5	7	10	13	15	18	21	23	26	Blank

2. concrete test procedure

A. 500 μ L sample diluting liquids are added the diluted sample pipe, add 5 μ L (1:101) reference substances then, calibration object and sample to be checked use pipettor to blow and beat (4～6 times) repeatedly, and be fully subsequent use behind the mixing.Negative control is prepared 2 parts, and strong positive contrast, calibration object and weak positive control are respectively prepared 3 parts;

B. according to shown in the above-mentioned application of sample table, every hole adds good each the 100 μ L of reference substance, calibration object and sample to be checked of dilution, covers the shrouding film, and 37 ℃ (± 1 ℃) are hatched 60min.The single hole check is all carried out in primary dcreening operation test, every duplicate samples, and the application of sample order is as shown in table 1.The reinspection of three holes is carried out in validation test, every duplicate samples, and the application of sample order is as shown in table 2.

C. discard liquid in each hole, use 96 holes to wash plate machine washing plate 4 times, add washing lotion 400 μ L/ holes at every turn, wash plate at every turn and left standstill 10 seconds, clap and do;

D. every hole adds the acid eluent of 200 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 15min (note: the time of this step is extremely important, must in strict accordance with requiring operation);

E. repeating step C;

F. every hole adds the enzyme conjugates of 100 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 30min;

G. repeating step C;

H. every hole adds 100 μ L colour developing liquid, puts 25 ℃ of (± 1 ℃) incubations 15 minutes;

J. every hole adds 100 μ L stop buffers, selects ELIASA wavelength 450nm, and reference wavelength 630nm measures each hole OD value.

[reference value (term of reference)]

2. select median (noting: be not mean value) as calibration object, the OD value of reference substance and sample to be checked (validation test).With the OD value of mean value as negative control.For example calibration object is 0.616,0.628,0.660, and median is 0.628 so, and then the OD value of this sample is 0.628.

3. the OD value scope of acceptable contrast and calibration object

The OD value	NC	CAL	LPC	HPC
					Minimum value	0.000	0.300	0.200	0.600
Maximal value	0.250	0.900	0.800	2.100

4.ODn the conversion method of value

5. the ODn value scope of acceptable contrast and calibration object

The ODn value	NC	CAL	LPC	HPC
					Minimum value	0.000	1.000	0.400	1.200

Maximal value

0.300

1.000

0.750

1.900

6. the OD value of each test and ODn value must be false otherwise test in above tolerance interval, must carry out repetition.

[explanation of test findings]

4. primary dcreening operation test: if the ODn of sample >=2.0, this sample is long-term infection sample, if sample ODn 2.0, then need carry out validation test (concrete operations see the validation test part for details);

5. validation test: < 2.0 sample need carry out validation test to primary dcreening operation test ODn.In the validation test, each sample carries out three holes to be rechecked, and validation test application of sample order is as shown in table 2, and concrete test procedure sees [test method] for details.If<1.0, this sample is the recent infection sample to the ODn of validation test sample, if sample ODn>=1.0 think that then this sample is the long-term sample that infects;

6. the result explains: the early-stage Study according to the scientist of U.S. Disease Control and Prevention Center shows that the CUTOFF value is that ODn=1.0 representes that the average positive commentaries on classics time is 141 days.But can distinguish to some extent in different regions, China still needs and will more test and data analysis the average positive commentaries on classics time.

[calculating of New Development infection rate]

By U.S. disease prevention and control center and global AIDS coordinating office, UNAIDS and World Health Organization's whole world AIDS and venereal disease working group combine recommendation, the computing formula of New Development infection rate that is used for the transversal section sample is following:

$I_F=\frac{R-(\mathrm{FRR}*P)}{(1-\mathrm{FRR})\mathrm{\&omega;N}}\&times;100$

I _FRepresent the New Development infection rate;

Negative sample quantity in the N representative investigation;

Positive sample size in the P representative investigation;

R represents the quantity that is judged as the sample that infects recently in the positive sample;

ω represents average window phase;

FRR represents false recent infection rate.

Can find out that from computing formula window phase ω and false recent infection rate FRR influence the whether accurate key influence factor of New Development infection rate estimation.Therefore, when using this formula calculating New Development infection rate, recommend following three preconditions:

A) there is suitable sample crowd to be used to estimate New Development infection rate and local FRR coefficient, obtain the FRR value of this area or own country, and this correction coefficient accurately and reliably;

B) there is the infected's of antiviral therapy history sample to foreclose, do not include the calculating of New Development infection rate in;

C) do not meet the sample of including testing conditions in, as far as possible deletion.

FRR coefficient calculations and the method that select to be suitable for sample cluster please refer to WHO governing principle ( Www.who.int/diagnostics_laboratory/hiv_incidence_may13_f inal.pdf).The FRR coefficient of China can be seeked advice from Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con's Reference Lab with the up-to-date information of average window phase.

The brainstrust of U.S. disease prevention and control center has been accomplished the Primary Study of U.S. FRR coefficient with average window phase at present, and particular content sees list of references [1] for details.

[limitation of the method for inspection]

This test only is applicable to monitoring crowd's HIV-1 New Development infection rate, can not be used for diagnosis of case.

List of references:

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6.Priddy?FH,Pilcher?CD,Moore?RH,et?al.Detection?of?acute?HIV?infections?in?an?urban?HIV?counseling?and?testing?population?in?the?United?States.J?Acquir?Immune?Defic?Syndr?2007,44:196-202.

7.Timothy?D.Mastro，MD，Andrea?A.Kim，PhD，MPH，Timothy?Hallett，PhD，et?al.Estimating?HIV?Incidence?in?Populations?Using?Tests?for?Recent?Infection：Issues，Challenges?and?the?Way?Forward.J?HIV?AIDS?Surveill?Epidemiol，2010January?1；2（1）：1-14.

8.Karita?E,Price?M,Hunter?E,Chomba?E,Allen?S,et?al.Investigating?the?utility?of?the?HIV-1?BED?capture?enzyme?immunoassay?using?cross-sectional?and?longitudinal?serocon-verter?specimens?from?Africa.AIDS?2007,21:403-408.

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Claims (9)

1. an I type human immunodeficiency virus (HIV-1) New Development infects the inspection-free test agent box of enzyme, it is characterized in that, comprises following component: HIV-1gp41 albumen; Blank elisa plate; Acid eluent; The goat anti-human igg of horseradish peroxidase-labeled; The TMB liquid that develops the color; Stop buffer.

2. according to the kit of claim 1, it is characterized in that wherein, HIV-1gp41 albumen is the gp41 recombinant protein; Blank elisa plate is the blank elisa plate of NUNC; Acid eluent is: 0.01M citrate buffer (pH=3.0); Enzyme conjugates is the enzyme conjugates dilution that contains the goat anti-human igg; The PBS damping fluid (pH=7.2) that wherein said enzyme conjugates dilution is 0.05M; Wherein contain 1% bovine serum albumin(BSA), 1% sucrose, 0.1% Tween-20 and 0.1% Triton-100; TMB colour developing liquid is the tetramethyl biphenyl amine aqueous solution; Stop buffer is the sulfuric acid solution of 1N.

3. according to the kit of claim 1, it is characterized in that, is that each component is prepared, and is mixed with the separately unit of packing that is fit to use, and the unit that comprises of the present invention is following:

① Encapsulate elisa plate ② 20 * lavation buffer solution ③ Sample diluting liquid ④ Acid eluent ⑤ Enzyme conjugates ⑥ Colour developing liquid ⑦ Stop buffer ⑧ Strong positive contrast (HPC) ⑨ Weak positive control (LPC) ⑦ Calibration object (CAL) ⑧ Negative control (NC)

4. according to the kit of claim 3, it is characterized in that the preparation method who wherein encapsulates elisa plate is following:

1) preparation technology

2) encapsulate: the pH that uses 0.05M is that 6.0 phosphate buffer is diluted to 0.063 μ g/mL with HIV-1gp41 albumen; Damping fluid after will diluting then joins in the microwell plate of 96T; Every hole adds 100 μ L, places 4 ° of C to encapsulate 18～22h microwell plate then;

3) wash plate: use robotization to wash plate machine washing plate 2 times, drain;

4) sealing: every hole adds 200 μ L confining liquids, then microwell plate is placed 4 ° of C sealing 18～22h;

5) drying: use robotization to wash the plate machine microwell plate is drained, dried overnight, drying condition is 18～25 ° of C of temperature, humidity < 30%;

6) pack: the elisa plate that will encapsulate is put into aluminium foil bag, encapsulates after putting into a drying agent, places under 4 ° of C conditions and preserves;

The prescription that encapsulates the various liquid that use in the process is following:

1.) the PBS damping fluid (pH=6.0) of coating buffer prescription: 0.05M;

2.) the PBS damping fluid (pH=6.0) of confining liquid prescription: 0.05M wherein contains 0.5% casein, 2% sucrose, 1% glycocoll, 0.1% Proclin-300;

3. the PBS damping fluid (pH=7.2) of) lotion prescription: 0.05M wherein contains 0.05% Tween-20; Pure water is all adopted in all liq preparation.

5. according to the kit of claim 1, it is characterized in that wherein, the preparation method of 20 * lavation buffer solution is following: the PBS damping fluid (pH=7.2) of 0.1M, wherein contain 0.1% Tween-20; The preparation method of sample diluting liquid is following: the PBS damping fluid (pH=6.0) of 0.05M, wherein contain 0.5% casein, 2% sucrose, 1% glycocoll and 0.1% Proclin-300; Acid eluent is 0.01M citrate buffer (pH=3.0); Enzyme conjugates is the enzyme conjugates dilution that contains the goat anti-human igg, and the PBS damping fluid (pH=7.2) that wherein said enzyme conjugates dilution is 0.05M wherein contains 1% bovine serum albumin(BSA), 1% sucrose, 0.1% Tween-20 and 0.1% Triton-100; The strong positive contrast is: the HIV-1 type positive serum of OD value scope between 0.6-2.1; Weak positive control is: the HIV-1 type positive serum of OD value scope between 0.2-0.8; Calibration object is: the HIV-1 type positive serum of OD value scope between 0.3-0.9; Negative control is: the HIV-1 type positive serum of OD value scope between 0.00-0.25.

6. according to the kit of claim 1, it is characterized in that, wherein, kit of the present invention, the loading amount of each component can confirm according to applicable cases, so that loading amount is appropriately for well, as is mixed with 96 person-portions, 192 person-portions, 480 person-portions etc.

7. according to the kit of claim 1, it is characterized in that wherein, 192 person-portions can be according to following method packing:

The component title Component concentration 20 * lavation buffer solution 60mL

Sample diluting liquid 60mL Acid eluent 60mL Enzyme conjugates 25mL Colour developing liquid 25mL Stop buffer 25mL Strong positive contrast (HPC) 50μL Weak positive control (LPC) 50μL Calibration object (CAL) ?50μL Negative control (NC) ?50μL

8. according to the kit of claim 1, it is characterized in that, wherein, each unit decals is known, be assembled into complete kit, be stored under the 2-8 ℃ of condition with-20 ° of C.

9. the method for application of the kit of claim 1 may further comprise the steps:

A. 500 μ L sample diluting liquids are added the diluted sample pipe, add 5 μ L (1:101) reference substances then, calibration object and sample to be checked use pipettor to blow and beat (4～6 times) repeatedly, and be fully subsequent use behind the mixing.Negative control is prepared 2 parts, and strong positive contrast, calibration object and weak positive control are respectively prepared 3 parts;

B. according to shown in the above-mentioned application of sample table, every hole adds good each the 100 μ L of reference substance, calibration object and sample to be checked of dilution, covers the shrouding film, and 37 ℃ (± 1 ℃) are hatched 60min.The single hole check is all carried out in primary dcreening operation test, every duplicate samples, and the application of sample order is as shown in table 1.The reinspection of three holes is carried out in validation test, every duplicate samples, and the application of sample order is as shown in table 2.

C. discard liquid in each hole, use 96 holes to wash plate machine washing plate 4 times, add washing lotion 400 μ L/ holes at every turn, wash plate at every turn and left standstill 10 seconds, clap and do;

D. every hole adds the acid eluent of 200 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 15min

E. repeating step C;

F. every hole adds the enzyme conjugates of 100 μ L, after the vibration, covers the shrouding film gently, puts 37 ℃ of incubation 30min;

G. repeating step C;

H. every hole adds 100 μ L colour developing liquid, puts 25 ℃ of (± 1 ℃) incubations 15 minutes;

J. every hole adds 100 μ L stop buffers, selects ELIASA wavelength 450nm, and reference wavelength 630nm measures each hole OD value.

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