CN110885829B - Diphtheria toxin aptamer DT05 and application thereof - Google Patents
Diphtheria toxin aptamer DT05 and application thereof Download PDFInfo
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Abstract
The invention relates to a diphtheria toxin aptamer DT05 and application thereof, wherein the sequence of the aptamer DT05 is as follows: 5'-GTCGCATGGAAGGAGCGACGAGGGACGGTCGCGCCGTGGCGCAGGCTAGGAGAGTACGCGCGGGACCGGCGGTCCAAGT-3'. The diphtheria toxin aptamer DT05 can be combined with diphtheria toxin with high affinity and high specificity, and can be used as an antagonist to effectively neutralize the toxic effect of diphtheria toxin and relieve the clinical symptoms of diphtheria toxin poisoning.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-affinity nucleic acid aptamer DT05 specifically combined with diphtheria toxin and application thereof.
Background
Diphtheria is a potentially fatal disease caused by infection with corynebacterium diphtheriae and its secreted potent Diphtheria Toxin (DT). Corynebacterium diphtheriae is a gram-positive bacterium, the causative agent of diphtheria. The toxic effects of diphtheria toxin are mainly mediated through ribosylation of adenosine diphosphate to inhibit elongation factor 2 (EF-2), and the toxic effects cause tissue necrosis of toxin production sites such as respiratory tract by forming pathogenic pharyngeal pseudomembranes and causing local airway edema. Hematogenous diphtheria toxin spreading can also cause cranial nerve dysfunction, peripheral neuropathy, and cardiotoxicity, which are responsible for 50-75% of diphtheria cases of death.
Although widespread vaccination with diphtheria toxoid vaccine has greatly reduced the incidence of diphtheria cases, the threat to diphtheria has not yet disappeared and diphtheria is still prevalent in some countries and regions. Periodic outbreaks of diphtheria have also occurred recently in the sea, nigeria, south africa, indonesia, laos, bangladesh, also in the manchurian and indonesia, with mortality rates as high as 10%. International travel and the migration of susceptible people also exacerbate the risk of diphtheria becoming a global disease. Therefore, the national quality supervision, inspection and quarantine general office of China issues notice and reminds for a plurality of times to prevent the multinational diphtheria epidemic situation from being transmitted into China. Therefore, the occurrence and treatment of diphtheria infection should not be cared for and should be prepared fully.
The most effective method for treating diphtheria is to inject Diphtheria Antitoxin (DAT) in time to neutralize diphtheria toxin and prevent further tissue injury, and to eliminate diphtheria bacillus and stop toxin production in combination with antibiotic treatment, so that the incidence rate and death rate of diphtheria can be greatly reduced. DAT is currently mainly a horse-derived diphtheria toxin antibody, and because of the risk of severe allergic reactions, there is a risk of developing serum diseases and certain zoonotics, many manufacturers have stopped production, and the global supply of DAT is extremely limited. Thus, in order to address the potential risk of diphtheria onset and to effectively treat diphtheria patients, there is a need for safer, more potent antagonists that antagonize the toxic effects of diphtheria toxin.
Aptamers are also known as "synthetic antibodies", "chemical antibodies", the chemical nature of which is the folding of a single-stranded oligonucleotide molecule (ssDNA or RNA) into a specific three-dimensional structure for high affinity and high specificity binding to a target substance. The aptamer was obtained by an in vitro screening procedure by systematic evolution of ligands by exponential enrichment (Systematic evolution of ligands by exponential enrichment, SELEX). The aptamer has the characteristics of high affinity, high specificity, in vitro synthesis, modification to change the function and pharmacokinetics characteristics, no immunogenicity, economy and the like. The aptamer drugs developed based on the above advantages can specifically block the biological functions of targets, for example, can be used as neutralizing antagonists of toxins, inhibitors of cytokines, tumor therapeutic drugs for blocking transcription factors, and the like. Therefore, screening out high specificity, high affinity diphtheria toxin binding aptamer as an antagonist of its toxic effects is of great scientific and clinical value.
Disclosure of Invention
It is an object of the present invention to provide a nucleic acid aptamer DT05 of diphtheria toxin with high specificity and high affinity; it is another object of the present invention to provide various applications of the aptamer DT05 in preparing a reagent for separating and enriching diphtheria toxin in a sample, preparing a reagent or kit for detecting diphtheria toxin, preparing a medicament for neutralizing or antagonizing diphtheria toxin, and the like.
The aim of the invention is realized by the following technical scheme: a diphtheria toxin aptamer DT05, the sequence of which is shown below:
5'-GTCGCATGGAAGGAGCGACGAGGGACGGTCGCGCCGTGGCGCAGGCTAGGAGAGTACGCGCGGGACCGGCGGTCCAAGT-3'(SEQ ID NO:1)。
the nucleic acid aptamer DT05 of the diphtheria toxin is obtained by an in vitro SELEX screening technology based on the nucleic acid aptamer, wherein carboxyl magnetic beads are used as a solid phase medium, the diphtheria toxin is used as a target, and the nucleic acid aptamer which is specifically combined with the diphtheria toxin is obtained by screening a ssDNA library through the diphtheria toxin magnetic beads, and is named as the nucleic acid aptamer DT05.
The nucleic acid aptamer DT05 of diphtheria toxin can be subjected to chemical modification such as fluorescent group, amino group, biotin, digoxin or polyethylene glycol at the 5 'end or the 3' end.
The aptamer DT05 of the diphtheria toxin has the effect of antagonizing the toxicity of the diphtheria toxin and can be used as a potential neutralizing antagonist of the diphtheria toxin. The application of the aptamer DT05 of diphtheria toxin in preparing medicines for neutralizing or antagonizing diphtheria toxin.
The application of the aptamer DT05 of the diphtheria toxin in preparing a reagent for separating and enriching diphtheria toxin in a sample.
Application of the nucleic acid aptamer DT05 of the diphtheria toxin in preparing diphtheria toxin detection reagent or kit.
The aptamer DT05 of the diphtheria toxin can also be applied to targeted treatment with the diphtheria toxin as an effector molecule.
Compared with the prior art, the invention has the advantages that:
1. the aptamer DT05 of the invention has no toxicity, small molecular weight, good permeability and easy synthesis and marking.
2. The synthesis cost of the aptamer DT05 is lower than that of antibody preparation, and the aptamer DT05 has short period and good reproducibility.
3. The aptamer DT05 of the present invention binds diphtheria toxin with high affinity and high specificity, has a dissociation constant of 103.2pM (95% IC:76.76-141.2 pM), and does not bind to other control proteins.
4. The aptamer DT05 of the invention has wide application prospect and important scientific and social value in the fields of diagnosis and treatment of diphtheria corynebacterium infection, diphtheria toxin mediated targeted treatment and the like, and particularly has the effect of antagonizing diphtheria toxin toxicity, and can be used as a potential neutralizing antagonist of diphtheria toxin.
Drawings
FIG. 1 is a diagram of bioinformatics simulation of the secondary structure of aptamer DT05.
FIG. 2 is a graph showing the specificity of the fluorescent binding rate assay for aptamer DT05. In fig. 2, the abscissa indicates the analyzed protein, and the ordinate indicates the fluorescence binding rate.
FIG. 3 is a plot of dissociation constants of the fluorescent binding rate assay for aptamer DT05 binding diphtheria toxin. The dissociation constant (Kd) was 103.2pM (95% IC:76.76-141.2 pM). In FIG. 3, the abscissa indicates the DNA concentration (pM), and the ordinate indicates the fluorescence binding rate.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples of the specification:
a diphtheria toxin aptamer DT05, which has the sequence:
5'-GTCGCATGGAAGGAGCGACGAGGGACGGTCGCGCCGTGGCGCAGGCTAGGAGAGTACGCGCGGGACCGGCGGTCCAAGT-3'(SEQ ID NO:1)。
the nucleic acid aptamer DT05 of diphtheria toxin is 100mM Na at 25 DEG C + ，1mM Mg 2+ The spatial structure is as follows:
the nucleic acid aptamer DT05 of the diphtheria toxin is subjected to chemical modification on the 5 'end or the 3' end of the nucleic acid aptamer DT05, including but not limited to fluorescent groups, amino groups, biotin, digoxin, polyethylene glycol and the like.
The nucleic acid aptamer DT05 of the diphtheria toxin is obtained by carrying out chemical modification on products obtained by carrying out truncated or prolonged or partial base substitution structural modification on the nucleic acid aptamer DT05, wherein the chemical modification comprises but is not limited to fluorescent groups, amino groups, biotin, digoxin, polyethylene glycol and the like.
The nucleic acid aptamer DT05 of the diphtheria toxin is obtained by an in vitro SELEX screening technology based on the nucleic acid aptamer, carboxyl magnetic beads are used as a solid phase medium, the diphtheria toxin is used as a target, and the nucleic acid aptamer specifically combined with the diphtheria toxin is obtained by screening a ssDNA library through the diphtheria toxin magnetic beads.
The screening method of the diphtheria toxin aptamer DT05 comprises the following steps:
(1) Preparation of screening library: a random ssDNA library shown in the following sequence was prepared:
5’-GTCGCATGGAAGGAGCGACGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGGGACCGGCGGTCCAAGT-3’；
(2) Coupling diphtheria toxin with carboxyl magnetic beads to prepare diphtheria toxin magnetic beads;
(3) Subjecting the ssDNA library to a heat activation treatment;
(4) Incubating the ssDNA library obtained in the step (3) with the diphtheria toxin magnetic beads obtained in the step (2);
(5) Magnetically separating the diphtheria toxin magnetic beads after the step (4), and washing off unbound, weakly bound and nonspecifically bound ssDNA on the surfaces of the diphtheria toxin magnetic beads; heating diphtheria toxin magnetic beads, and collecting ssDNA specifically bound with the diphtheria toxin magnetic beads, namely ssDNA enrichment library;
(6) And (3) PCR amplification: performing PCR amplification on the ssDNA enrichment library obtained in the step (5), wherein the primers used for the PCR amplification are as follows:
primer DTup:5'-FAM-GTCGCATGGAAGGAGCGACG-3'
Primer DTdown:5'-Biotin-ACTTGGACCGCCGGTCCCG-3';
(7) Purification of PCR products: purifying the PCR product by using a small fragment DNA purification kit; incubating the purified dsDNA with streptavidin magnetic beads, washing the streptavidin magnetic beads combined with the dsDNA, melting the dsDNA, separating by using a magnetic frame, and collecting the supernatant; the supernatant is subjected to ethanol precipitation to obtain a secondary ssDNA library for the next round of screening;
(8) And (3) circularly screening: taking the FAM-labeled secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of the steps (3) to (7).
Embodiment one: screening of aptamer DT05
The screening method of the diphtheria toxin aptamer DT05 comprises the following steps:
(1) Preparation of screening library: designing a ssDNA random library, wherein the sequence of the ssDNA random library is as follows:
5'-GTCGCATGGAAGGAGCGACGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGGGACCGGCGGTCCAAGT-3' comprising a fixed sequence region at both ends (20 nucleotides at the 5 'end and 19 nucleotides at the 3' end) and a random sequence region in the middle (40 random sequence nucleotides) and was assigned to the engineering company, inc.
(2) Diphtheria toxin is coupled with carboxyl magnetic beads: the diphtheria toxin is derived from a corynebacterium diphtheriae NCTC10648 strain with purity of >98%, and is purchased from the company The Native Antigen Company, and the carboxyl magnetic beads and the coupling reagent are purchased from the company Bangs Laboratories. Diphtheria toxin coupled to magnetic beads procedure is referred to the manufacturer's instructions. The protein concentration change in the diphtheria toxin solution before and after coupling is measured by the protein concentration BCA method, and the coupling efficiency of the magnetic beads is calculated to be 83%; diphtheria toxin magnetic beads were dispersed in PBS buffer and stored at 4 ℃.
(3) 1nmol of ssDNA random library was dissolved in 500. Mu.L of selection buffer (50 mM Tris-HCl,100mM NaCl,1mM MgCl) 2 5mM KCl, pH 7.4) and then heat activated. The method for heat activation treatment comprises the following steps: denaturation at 95℃5Immediately after the min, the mixture was placed in an ice water bath for 10min and then at room temperature for 10min.
(4) The ssDNA library after step (3) was incubated with the diphtheria toxin beads (diphtheria toxin loading 20 ng) obtained in step (2) for 1h at room temperature with yeast tRNA (molar amount 5 times that of ssDNA library).
(5) Magnetically separating the diphtheria toxin magnetic beads after the step (4), and washing off unbound, weakly bound and nonspecifically bound ssDNA on the surfaces of the diphtheria toxin magnetic beads by using a selection buffer containing 0.2% BSA; then the diphtheria toxin beads were treated with 200. Mu.L ddH 2 O is resuspended, and after 5min of hot water bath at 100 ℃, the mixture is placed in a magnetic rack for 1-2min, and supernatant is collected to obtain ssDNA which is specifically combined with diphtheria toxin magnetic beads, namely ssDNA enrichment library.
(6) And (3) PCR amplification: adding the ssDNA enriched library obtained in step (5) to 1mL PCRmix; after vortex shaking and mixing, 50 mu L of each tube is subpackaged for PCR amplification, and the amplification conditions are as follows: pre-denaturing at 94 deg.c for 5 min; denaturation at 94℃for 30S, annealing at 63℃for 30S, elongation at 72℃for 30S,15-25 cycles.
Wherein 1mL of PCRmix contains: 100. Mu.L of 10 XPCR buffer; pfu enzyme 3. Mu.L; dNTP 20. Mu.L; primer DTup:5'-FAM-GTCGCATGGAAGGAGCGACG-3' and primer DTdown: 3 mu L each of 5'-Biotin-ACTTGGACCGCCGGTCCCG-3'; the primer DTup and the primer DTdown are both consigned to be synthesized by the engineering and bioengineering Co.Ltd.
(7) Purification of PCR products: PCR products, labeled with biotin and fluorescent groups FAM at both ends, were purified using a small fragment purification kit (the small fragment purification kit was purchased from Biotechnology Co., ltd.), the purified dsDNA was incubated with streptavidin beads (purchased from Invitrogen-Dynal Co.) at 37℃for 20min, and after washing the dsDNA-binding streptavidin beads three times with wash buffer (5 mM Tris-HCl, pH 7.5,1M NaCl, 500. Mu.M EDTA), dsDNA was melted by incubation with 50. Mu.L NaOH solution (0.1M) for 30min at 37 ℃; the FAM-labeled secondary ssDNA library was obtained by ethanol precipitation of the supernatant, which was isolated with a magnetic rack, and dissolved in selection buffer as the secondary library for the next round of screening.
(8) The screening process was performed for a total of 12 rounds. From the second round, the secondary libraries were used in an amount of 30pmol each.
Embodiment two: acquisition and analysis of nucleic acid aptamer DT05 sequence:
(1) After 12 rounds of screening, the enriched ssDNA library was collected and the beijing xinnaobao medical examination was committed to all companies to analyze the library sequence using high throughput sequencing technology, the analysis process was: PCR amplifying the enriched library and adding sequencing adapter and Index portions; selecting a purified library by gel electrophoresis; measuring the concentration and purity of the DNA by using the Nanodrop one for quality control analysis; using Illumina NovaSeq TM 6000 platform, using single-chain library as template to make bridge PCR amplification, sequencing primer annealing and sequencing while synthesizing; and comparing and enriching the sequencing result.
(2) According to the enrichment degree of the aptamer in the library, ssDNA with high enrichment degree is selected as a candidate aptamer, wherein the aptamer DT05 accounts for 13.5% of the enrichment library, and the sequence of the aptamer DT05 is shown as SEQ ID NO. 1.
(3) Analysis using a UNAFold network platform at 25℃with 100mM Na + ，1mM Mg 2+ Under the conditions of the nucleic acid aptamer DT05 sequence. The secondary structure schematic diagram of the sequence of the aptamer DT05 is shown in FIG. 1.
Embodiment III: specificity analysis of aptamer DT 05:
(1) FAM-labeled aptamer DT05 was synthesized chemically in vitro and dissolved in selection buffer.
(2) Referring to step (2) of example one, BSA (purchased from Sigma), pertussis Toxin (PTX, purchased from UK The Native Antigen Company) and Staphylococcus aureus enterotoxin B (SEB, purchased from Toxin Technology Co. U.S.A.) were coupled to carboxyl magnetic beads to prepare BSA magnetic beads, PTX magnetic beads and SEB magnetic beads, respectively.
(3) Mixing 200 mu L of the aptamer DT05 solution obtained in the step (1) with the BSA magnetic beads, the PTX magnetic beads, the SEB magnetic beads and the diphtheria toxin magnetic beads prepared in the step (2), incubating for 1h in a cassette at room temperature, and taking blank magnetic beads as blank control.
(4) Washing the magnetic beads of step (3) with 0.1% PBST for 3 times, and eluting the nucleic acid aptamer bound to the magnetic beads by boiling with 200. Mu.L selection buffer at 100℃for 5 min.
(5) The fluorescence intensities of the initial solution and the eluent are respectively measured by a fluorescence quantitative instrument, and the fluorescence binding rate = (initial fluorescence intensity-elution fluorescence intensity)/initial fluorescence intensity x 100% is calculated, and the calculated value primarily represents the binding rate of the nucleic acid aptamer DT05 and the target molecule.
As shown in FIG. 2, the binding rate of the aptamer DT05 and diphtheria toxin is obviously higher than that of the aptamer DT05 and BSA, PTX, SEB, which indicates that the aptamer DT05 and diphtheria toxin have better specificity.
Embodiment four: affinity analysis of aptamer DT05
(1) Mixing FAM-labeled aptamer DT05 solutions with different concentrations with diphtheria toxin magnetic beads, and incubating in a cassette at room temperature for 1h.
(2) Referring to the step (4) and the step (5) in the third embodiment, the fluorescence binding rate of the nucleic acid aptamer DT05 solution with different concentrations and the diphtheria toxin magnetic beads is obtained and calculated through experiments.
(3) And drawing a saturated binding curve of the nucleic acid aptamer DT05 to the diphtheria toxin by using the calculated value of the fluorescence binding rate, and calculating the dissociation constant of the nucleic acid aptamer DT05 to the diphtheria toxin by nonlinear regression analysis.
As shown in FIG. 3, the saturated binding curve of the aptamer DT05 is obtained, and the dissociation constant of the aptamer DT05 is 103.2pM (95% IC:76.76-141.2 pM) calculated, which shows that the aptamer DT05 has strong binding capacity to diphtheria toxin and is in picomolar level.
Fifth embodiment: research on antagonism of toxic effects of diphtheria toxin by aptamer DT05
(1) Determining the diphtheria toxin test dose: diphtheria toxin test dose is determined according to the standard method recommended by the national institutes of health, the main method being as follows: guinea pigs weighing around 250g (purchased from the 900 st hospital animal center) were purchased, and after 1d adaptation to the test environment, were grouped according to diphtheria toxin dose, 5 Diphtheria Antitoxin (DAT) per group, 1IU of diphtheria antitoxin (DT) was mixed with different doses of Diphtheria Toxin (DT) to prepare a mixture (designated DAT-DT), and incubated at room temperature for at least 1h. DAT-DT was subcutaneously injected at 0d per animal in a volume of 3.0mL. The physical condition and mortality of guinea pigs were observed, and the dose of diphtheria toxin, which all dead guinea pigs within 96 hours, was taken as the test dose. The lowest lethal dose was observed to be chosen as the test dose, namely ≡4fl (4.0156 fL).
(2) Method for studying the antagonism of the toxic effects of diphtheria toxin by aptamer DT 05:
A. preparation of polyethylene glycol aptamer DT 05: the method comprises the steps of synthesizing a nucleic acid aptamer DT05 with amino modification at the 5 'end and dT modification at the 3' end by the principal bioengineering Co-Ltd; and then, carrying out polyethylene glycol treatment on the modified nucleic acid aptamer DT05 by using 40-kDa polyethylene glycol (PEG), thus obtaining the polyethylene glycol nucleic acid aptamer DT05 (PEG-DT 05), and carrying out HPLC homozygosity on the PEG-DT05 for later use by entrusting the polyethylene glycol process to the Beijing key Kai technology Co., ltd.
B. Animal model experiment of aptamer DT 05: guinea pigs weighing around 250g (purchased from the 900 st hospital animal center) were purchased, and after 1d adaptation to the test environment, 5 animals per group were grouped according to doses of diphtheria antitoxin and aptamer, and different doses of pegylated aptamer DT05 (PEG-DT 05), diphtheria Antitoxin (DAT) were mixed with 4fL Diphtheria Toxin (DT) to prepare mixtures (designated PEG-DT05-DT and DAT-DT, respectively) and incubated at room temperature for at least 1h. PEG-DT05-DT and DAT-DT were subcutaneously injected at 0d per animal, in a volume of 3.0mL. The physical condition and mortality of guinea pigs were observed, and the observation time was extended from standard 96h to 30d to allow greater discrimination of dose effects and assessment of signs of toxin-induced end organ damage occurring 30d after toxin exposure. Animals were observed daily for signs of diphtheria toxin poisoning, and body weight was measured weekly as a measure of overall health. Establishing a digital scoring system for clinical symptoms: asymptomatic = 0; sloppy fur = 1; comatose = 2; dehydration = 3; dragging one or two hind legs = 4; moribund = 5; death=6. Animals that are dying or animals that have a weight loss of 20% from baseline are euthanized.
(3) Results of studies on antagonism of diphtheria toxin toxic effects by aptamer DT 05: as shown in Table 1, in the animal model receiving DAT-DT injections, all guinea pigs receiving DAT doses of 1.25IU or less died, and the lower the DAT dose, the earlier the guinea pigs died; all guinea pigs receiving a DAT dose of > 1.75IU survived. Survival was variable in guinea pigs receiving DAT doses of 1.5IU-1.6 IU. In the animal model with PEG-DT05-DT injection, all guinea pigs receiving the PEG-DT05 dose of 16 μg or less die, and the lower the dose, the earlier the guinea pigs die; all guinea pigs receiving a PEG-DT05 dose of > 64 μg survived. Survival was variable in guinea pigs receiving PEG-DT05 doses of 16-64 μg, suggesting that the aptamer DT05 acts similarly to diphtheria antitoxin. In addition, clinical symptoms of the tested guinea pigs are observed, and when the dosage of the PEG-DT05 is more than or equal to 80 mug, the clinical manifestation fraction of all the guinea pigs is less than or equal to 3, which proves that the aptamer DT05 can be used as an antagonist to effectively neutralize the toxic effect of diphtheria toxin and relieve the clinical symptoms of diphtheria toxin poisoning.
TABLE 1 protection of diphtheria toxin lethal guinea pig model by aptamer DT05 and diphtheria antitoxin
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that changes, modifications and adaptations to those skilled in the art may be made without departing from the spirit of the present invention and are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Changle district Baoaking winter medical technology Co., ltd
<120> nucleic acid aptamer DT05 of diphtheria toxin and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 79
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
gtcgcatgga aggagcgacg agggacggtc gcgccgtggc gcaggctagg agagtacgcg 60
cgggaccggc ggtccaagt 79
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gtcgcatgga aggagcgacg 20
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
acttggaccg ccggtcccg 19
Claims (5)
1. A diphtheria toxin aptamer DT05, characterized by:
its sequence is shown in SEQ ID NO:1 is shown in the specification; and at 25 ℃,100mM Na + ，1mM Mg 2+ The spatial structure of it is as follows:
2. the diphtheria toxin aptamer DT05 according to claim 1, wherein: and (3) carrying out chemical modification on a fluorescent group, an amino group, biotin, digoxin or polyethylene glycol on the 5 'end or the 3' end of the aptamer DT05.
3. Use of the aptamer DT05 to diphtheria toxin according to claim 1 for preparing an isolating and enriching reagent for diphtheria toxin in a sample.
4. Use of the aptamer DT05 to diphtheria toxin according to claim 1 for the preparation of a diphtheria toxin detection reagent or kit.
5. Use of the aptamer DT05 to diphtheria toxin according to claim 1 for the preparation of a medicament for neutralizing or antagonizing diphtheria toxin.
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