Detection of antigens

Abstract

The invention discloses a method for detecting at least one antigen, comprising the following steps: providing magnetic beads, which are coated with antibodies specific for at least one antigen to be detected; bringing the magnetic beads in contact with a washing buffer that comprises at least 8% BSA and incubating the mixture with a sample; isolating the magnetic beads by means of magnetic separator; and detecting the antigens bound to the magnetic beads by the antibodies. Washing buffers, primers, kits and devices that can be used for said methods are also disclosed.

Claims

1. A method for the detection of at least one antigen in a sample comprising the steps of: (a) coating magnetic beads with at least one antibody specific for the at least one antigen; (b) washing the coated beads with a first washing solution comprising a buffer and about 8% to 12% bovine serum albumin (BSA); (c) incubating the washed beads with the sample to form a mixture; (d) washing the mixture with a second washing solution comprising a buffer; (e) isolating the beads using a magnetic separator; and (f) detecting the at least one antigen in the sample.

2. The method of claim 1 wherein the first washing solution further comprises a detergent.

3. The method of claim 1 wherein the second washing solution further comprises about 8% to 12% BSA.

4. The method of claim 1 wherein the first washing solution and the second washing solution are the same.

5. The method of claim 1 wherein the washing step (b) is performed by incubating the coated beads in the first washing solution for at least 30 minutes to 12 hours.

6. The method of claim 1 wherein detecting the at least one antigen is performed using a nucleic acid amplification method.

7. The method of claim 1 wherein detecting the at least one antigen is performed using an enzyme-linked immunosorbent assay (ELISA) assay.

8. The method of claim 1 where the washing step (b) is repeated at least one time prior to the incubation step (c).

9. The method of claim 1 wherein the washing step (d) is repeated at least one time after the incubation step (c).

10. The method of claim 1 wherein the washing step (d) occurs on a magnetic separator.

11. The method of claim 1 where the at least one antigen to be detected is from a microorganism selected from the group consisting of a bacterium, a virus, a fungus, a toxin, LPS, and protozoa.

12. The method of claim 1 where the at least one antibody comprises an antibody specific for a microorganism selected from the group consisting of a bacterium, a virus, a protozoon, a fungus; and an antibody specific for a toxin produced by the microorganism, the bacterium, the virus, the protozoon, and the fungus.

13. The method of claim 11 wherein the microorganism is selected from Escherichia coli, adenovirus, and Legionella spp.

14. The method of claim 12 wherein the microorganism is selected from Escherichia coli, adenovirus, and Legionella spp.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the results of an experiment for blocking the unspecific binding sites of the beads.

(2) FIG. 2 shows the detection of Legionella pneumophila by means of an immunological method according to the invention.

(3) FIG. 3 shows the detection of adenovirus by means of an immunological method according to the invention.

(4) FIG. 4 shows the detection of E. coli by means of an immunological method according to the invention.

(5) FIG. 5 shows the qualitative and quantitative detection of Legionella pneumophila by means of a nucleic acid amplification method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

Example 1

Production of the Antibody-Coated Beads

(6) For the following detection experiments of antigens using the method of the invention, streptavidin beads and the following primary and secondary IgG antibodies or antibody pairs, respectively, have been used:

(7) TABLE-US-00001 Antibody Organism Company anti-Adenovirus, goat, polyclonal Acris Antibodies biotin-conjugated anti-Adenovirus, goat, polyclonal Acris Antibodies HRP-conjugated anti-E. coli, biotin-conjugated rabbit, polyclonal Novus Biologicals anti-E. coli, HRP-conjugated rabbit, polyclonal Novus Biologicals anti-L. pneumophila rabbit, polyclonal Acris Antibodies anti-L. pneumophila, rabbit, polyclonal Acris Antibodies biotin-conjugated control antibodies employed: anti-goat IgG, HRP-conjugated rabbit, polyclonal Abcam anti-rabbit IgG, HRP-conjugated goat Novus Biologicals

(8) For producing the antibody-coated beads, the streptavidin beads were incubated with one of the biotin-conjugated antibodies, respectively.

Example 2

Optimisation of the Washing Buffer

(9) A component of the washing buffer that is essential for the invention is the BSA present at a certain concentration. For determining the optimal concentration of BSA for the method of the invention, test series were conducted, wherein an unspecific binding of adenoviruses to the beads themselves was examined. For this, the beads were incubated overnight with different blocking reagents (0.1% BSA; 10% BSA; 5% milk powder, and 0.5 mg/ml biotin). In parallel, control experiments were performed to exclude that the blocking reagents employed inhibit the interaction between antigen and antibody. The beads were blocked with the washing buffer of different BSA concentrations and after incubation with the adenoviruses were washed with the same buffer before an adenovirus-specific PCR was performed.

(10) FIG. 1 shows the results of this experiment of blocking unspecific binding sites of the beads with subsequent adenovirus-specific PCR. In particular, lane 1) shows DNA marker and lanes 2-5 positive controls, wherein lane 2) shows beads coated with adenovirus-specific antibodies, 0.1% BSA in the washing buffer, and incubation with adenoviruses, lane 3) shows beads coated with adenovirus-specific antibodies, 10% BSA in the washing buffer, and incubation with adenoviruses, lane 4) shows beads coated with adenovirus-specific antibodies, 5% milk powder in the washing buffer, and incubation with adenoviruses, lane 5) shows beads coated with adenovirus-specific antibodies, 0.5 mg/ml biotin in the washing buffer, and incubation with adenoviruses. Lane 6) is empty.

(11) The results of the blocking assays can be seen in lanes 7-10, wherein lane 7) shows uncoated beads with 0.1% BSA in the washing buffer and incubation with adenoviruses; lane 8) shows beads with 10% BSA in the washing buffer and incubation with adenoviruses; lane 9) shows beads with 5% milk powder in the washing buffer and incubation with adenoviruses; lane 10) shows beads with 0.5 mg/ml biotin in the washing buffer and incubation with adenoviruses.

(12) As a result, it can clearly be seen that with insufficient blocking, adenoviruses bind to beads unspecifically and the corresponding genome section is amplified in the subsequent PCR (lanes 7 and 10). With sufficient blocking, the adenoviruses are not bound unspecifically and no amplification takes place.

(13) The experiment has been repeated with Legionella and with comparable experimental results.

(14) Taken together, it could be shown that with a lower concentration of BSA (e.g. 0.1%), the antigen (Legionella or Adenoviruses) binds unspecifically to the magnetic beads and the desired specific antigen-antibody reaction of the invention does not occur.

(15) It could be shown experimentally that blocking (saturating unused binding sites on the beads) before and during the washing can massively prevent an unspecific interaction. The blocking of the beads with 10% BSA overnight yielded the best results. A blocking of the beads with 8% BSA also yielded very good results. For this reason, BSA in a range of 3-12% was added to the washing buffer (PBS).

Example 3

Immunological Detection

(16) The method of the invention was performed with subsequent immunological detection.

(17) For this, the antibody pairs listed above were used, as well as the following solutions:

(18) PBS Washing Buffer:

(19) TABLE-US-00002 Final Amount concentration Potassium chloride 0.2 g 2.7 mM Potassium dihydrogen phosphate 0.2 g 1.5 mM Disodium hydrogen phosphate 1.15 g 8.1 mM Sodium chloride 8.0 g 136.9 mM BSA, biotin-free 100 g 8% Tween 20 0.1 ml 0.01% Aqua dest. ad 1000 ml pH 7.4 adjusted with hydrochloric acid or sodium hydroxide.
Conjugate Solution

(20) The conjugated antibody was diluted in PBS 1:750.

(21) HRP Buffer

(22) TABLE-US-00003 Potassium citrate 6.9 g 30 mM Aqua dest. ad 1000 ml Adjust to pH 4.1 with potassium hydroxide
TMB Substrate Solution

(23) TABLE-US-00004 Tetramethylbenzidine 240 mg 10 mM Acetone (96%) 10 ml Ethanol (96%) 90 ml Hydrogen peroxide (30%) 750 l
Stopping Solution

(24) TABLE-US-00005 96% sulphuric acid 6 ml 1M Aqua dest. ad 95 ml

(25) The method was performed according to the following schedule: 1) Withdrawal of 8 l of antibody-coated beads (7-1210.sup.8 beads/ml) and transfer into the reaction tube 2) 5 washing of the beads on the magnetic separator with 400 l PBS at a time 3) Uptake into 8 l PBS 4) Addition of 36 l sample 5) Incubation of 1 h at room temperature, stirring of the sample every 15 min 6) 5 washing of the beads on the magnetic separator with 400 l PBS at a time 7) Uptake into 200 l conjugate solution 8) Incubation of 1 h at room temperature, stirring of the sample every 15 min 9) 5 washing of the beads on the magnetic separator with 400 l PBS at a time 10) Uptake of the beads into 20 l PBS 11) Pipetting 1000 l HRP buffer+50 l TMB substrate solution into a reaction tube and addition of the 20 l beads from step 10 12) Stirring of the sample 13) Incubation of 45 min at room temperature 14) Stopping of the reaction with 500 l stopping solution 15) Stirring of the sample 16) Incubation of 10 min at room temperature 17) Evaluation of the assay using the evaluation instrument

(26) FIG. 2 shows the results of the detection of Legionella pneumophila, wherein different Legionella dilutions were detected using the method described above. The detection limit lay at a dilution of 1:10,000, corresponding to a total cell number of 7,416 Legionella.

(27) FIG. 3 shows the results of the detection of Adenovirus (subgroup C, serotype 6), wherein the adenoviruses were diluted by means of a dilution series and incubated with the beads coated with specific antibodies. The actual detection occurred subsequently also via the conjugate using the enzymatic reaction of the horseradish peroxidise (HRP). Adenoviruses could be specifically detected down to a dilution of 1:100.

(28) FIG. 4 shows the results of the detection of E. coli, wherein the bacteria were diluted in steps of 10 and incubated with magnetic beads, respectively. After stringent washing, the bacteria were also detected colorimetrically with TMB as substrate. E. coli could be detected down to a dilution of 1:100, corresponding to a total cell number of 6,000 bacteria (after cultivation of the bacteria on Petri dishes, a cell number of 6,000 organisms could be determined for the 1:100 dilution).

Example 4

Detection Using Nucleic Acid Amplification (PCR)

(29) Besides the immunological detection of Legionella pneumophila and Adenovirus, molecular biological assays using the PCR methods were also developed. The method of the invention was thus performed with subsequent detection via PCR.

(30) For this, the antibodies mentioned above were used for the production of the coated beads, as well as the following solutions:

(31) PBS Washing Buffer:

(32) TABLE-US-00006 Final Amount concentration Potassium chloride 0.2 g 2.7 mM Potassium dihydrogen phosphate 0.2 g 1.5 mM Disodium hydrogen phosphate 1.15 g 8.1 mM Sodium chloride 8.0 g 136.9 mM BSA, biotin-free 100 g 10% Tween 20 0.1 ml 0.01% Aqua dest. ad 1000 ml pH 7.4 adjusted with hydrochloric acid or sodium hydroxide.
PCR Reaction Buffer with MgCl.sub.2

(33) 10 mM Tris-HCl (pH 8.3),

(34) 50 mM KCl

(35) 1.5 mM MgCl.sub.2

(36) Nucleotide Mix

(37) A final concentration of 200 M nucleotide mix was used per assay. The stock solution amounted to 10 mM.

(38) Taq DNA Polymerase

(39) 0.02 units/l Taq DNA polymerase were used per assay.

(40) The following primer pairs were used for the specific detection of Legionella pneumophila and Adenovirus:

(41) TABLE-US-00007 Primersequence Legionellapneumophila: ompS_Leg_forward 5 -GCGGCTGTATTTGCTCTGGGAA-3 ompS_Leg_reverse 5 -TAAGCCTATGTAGGGGCCAGATGC-3 Adenovirus: hexon_AdV_forward 5 -GAAATGACACCAACGACCAG-3 hexon_AdV_reverse 5 -GGGAACATTAGCGGGGTAAG-3

(42) 100 pmol per primer were used per assay.

(43) These primer pairs are specific for certain marker genes in Legionella and Adenovirus.

(44) The detection of Legionella pneumophila was respectively performed with specific primers for the gene ompS, encoding a highly conservative membrane protein. The primers of this primer pair lie at positions 294-315 and 1119-1142 of the DNA sequence of the ompS protein of L. pneumophila, which is known to the skilled person (Gene Bank accession number: M76178.1).

(45) The detection of Adenovirus was respectively performed with specific primers for the capsid gene hexon. Here the primer pair was designed based on conserved regions. The primers of this primer pair lie at positions 1937-1956 and 2555-2574 of the DNA sequence of the hexon protein of adenoviruses, which is known to the skilled person (Gene Bank accession number: AB330087.1).

(46) For the amplification of DNA fragments of both micro-organisms, one corresponding primer pair, of those listed above was used, respectively. The amplification occurred in several cycles with three reaction steps each. In the first reaction step, the DNA double strand was melted into single strands by heating to 95 C., which the complementary oligonucleotides annealed to upon quick chilling. In the third reaction step, starting from the primers, double strands were synthesised in the presence of the nucleotides (dNTPs), while the temperature was increased to 72 C., the optimal reaction temperature of the polymerase.

(47) The amplification occurred under the following conditions:

(48) TABLE-US-00008

(49) At the beginning of the amplification round, the matrix DNA was heated for 5 min to 95 C. in order to denature it completely. Afterwards, the amplification of the DNA fragment occurred in 30-35 cycles. As annealing temperature, the value of the primer having the lower melting temperature was used. The polymerisation time was chosen between 20 s and 3 min, depending on the length of the fragment to be amplified. A polymerisation period of 1 min per 1 kb was assumed. After the last polymerisation step at 72 C., this temperature was maintained for further 5 min in order to ensure complete polymerisation. 50 l mixtures in 0.5 ml reaction tubes were prepared.

(50) Experimental Results for Legionella pneumophila

(51) Experiments for the qualitative and quantitative detection using the marker gene ompS were performed. For the detection of Legionella pneumophila via PCR, 5 l beads were used, respectively, to which Legionella from a dilution series had previously been bound. The detection occurred with specific ompS primers, respectively. Beads without template, beads+PBS, and beads with a foreign antigen were used as negative controls, respectively.

(52) As can be recognised in FIG. 5, it could be shown that only beads with bound Legionella as template yielded a signal (qualitative detection). For the quantification, a PCR on genomic Legionella DNA was performed simultaneously. For this, a defined amount of genome copies (210.sup.6) was diluted in a dilution series and employed in the PCR. After electrophoretic separation of the amplificates the detection limit could be estimated by means of the agarose gel (quantitative detection).

(53) FIG. 5 shows the qualitative and quantitative detection of Legionella pneumophila.

(54) On the upper agarose gel, a dilution series starting from a defined genome copy concentration can be seen: lane 1) 210.sup.6 genomes, lane 2) 210.sup.5 genomes, lane 3) 210.sup.4 genomes, lane 4) 210.sup.3 genomes, lane 5) 210.sup.2 genomes, lane 6) 210.sup.1 genomes, lane 7) negative control without genome copies.

(55) On the lower agarose gel, a dilution series of Legionella pneumophila can be seen: lane 8) detection of about 210.sup.3 Legionella bound to magnetic beads, lane 9) detection of about 210.sup.1 Legionella bound to magnetic beads, lane 10) detection of about 210.sup.2 Legionella bound to magnetic beads, lane 11) detection of about 210.sup.0 Legionella bound to magnetic beads, lane 12) negative control without Legionella bound to magnetic beads, lane 13) PCR with washing buffer (PBS), lane 14) PCR with foreign antigen, lane M) DNA marker a band corresponds to 100 base pairs.

(56) As can further be derived from FIG. 5, the detection limit lies at a concentration of 2,000 to 200 cells/12 l.

(57) Experimental Results for Adenoviruses

(58) For the detection of Adenoviruses, the above-mentioned primers for the marker gene hexon were used. It could be shown that results comparable to the Legionella can be achieved with these primers.