Developing Biological Agent Detection System

by Koji OSHIMA
Senior Research Councilor, DRC

Foreword

Last year I briefly described biological weapons in the 21st century. (DRC Annual Report, No4, September 2000) The Defense Agency and the defense forces have never seriously studied on protection against biological agents. The first step of protection against the agents is rapid and exact detection of the agents. Here I will briefly discuss how to develop a biological agent detection system in Japan.

1. Use of Biological Agent

Biological agents have characteristics of ①attacking only living organisms, ②having a latent period, ③multiplying themselves, ④having effects heavily dependent upon meteorology, ⑤difficulty in controlling effects. Because of these characteristics biological agents are mainly used against strategic targets in a rear area secretly, or used in a low-intensity conflict such as a sabotage, guerilla, and terror.

(1) Targets

Strategic targets are supposed to be ①local people in a build-up area, ②an critical industrial complex, ③the national command and control center, and ④an important rear area facility such as an airport and a military port. And targets in a low-intensity conflict will be ①specific VIPs’, ②the official residence of the prime minister and/or the government office buildings, ③facilities of the national power structure such as the Self Defense Forces, the police and U.S. Military forces, ④public facilities such as an airport and a railroad station where many people gather at the same time, ⑤a lifeline center such as a filtration plant and a distribution reservoir.

(2) Attacking Measures

Strategic attacking Means will be ①a bomb, a free rocket and a missile, ②spray from an aircraft, ③a special aerosol generator. A special design is needed to disperse biological agents from a bomb, a free rocket and/or a missile in order to increase their survivability. Examples are ①an unglazed pottery bomb with a parachute attached, ②an aerosol generating bomblet with a parachute, which uses the Venturi Effect caused by the air flow while dropping, ③an aerosol generating bomblet with a propeller, ④a bomb filled with contaminated feathers, tree leaves or hard cards, ⑤a bomb filled with vectors such as flies, mosquitoes or fleas. When a biological agent is dispersed as an aerosol, it may be encapsulated in a microcapsule in order to prevent it from drying up by the environment. The particles in the aerosol will be controlled between 1-100μm, especially 1-10μm in size, which is suitable to invade into the respiratory track and to adhere the surface of the nostrils and the lungs.

In a low-intensity conflict, a special device will be used; a pen-shaped or umbrella-shaped sabotage device, a glass-made ball filled with a biological agent, a simple spraying device made of a spraying can, gel containing a biological agent.

Among the attacks above an attack by aerosol is the most threatening and the next one is a water contamination. Therefore we must be prepared for detecting both two threats.

2. Requirement for Biological Agent Detection System

(1) Purposes of Biological Agent Detection

Purposes of the detection are ①avoidance of contamination, ②appropriate and timely protection, ③proper and timely decontamination, ④adequate evacuation and treatment of casualties. Avoidance of contamination includes predicting and avoiding a drift area of a biological agent cloud. Appropriate and timely protection includes timely start or stop of a collective protection system, timely and proper wear and removal of an individual protection gear, and timely and proper administration of a medical protective means. Decontaminating area must be determined in order to decontaminate properly and timely, and afterward the result of the decontamination must be checked. Before evacuation and treatment of casualties a guideline must be established.

In a low-intensity conflict a primitive damage from an attack usually cannot be avoided. Hence it is necessary to avoid the secondary damage and prevent spread of the contamination after the attack is detected. Then detection of a biological agent enables ①to determine an exact contaminated area and concentration, ②to prevent spread of a contamination, ③to decontaminate adequately and timely, ④to evacuate and treat casualties adequately and timely.

(2) Details of Detection

In order to accomplish those purposes above, following factors are to be detected;①continuous monitoring and predicting drift area of a biological agent aerosol, ② measuring concentration of a biological agent at many points and predicting contaminated area to be avoided. Capability of identifying a type of a biological agent is better, but physical protections among different types of biological agents are the same. Although medical preventives among different types of agents differ, all agents have respective latent times and we have enough time to respond after arrival of an agent when we can identify a type of an agent. For decontamination, only detecting existence of an agent is necessary. For evacuation and treatment of casualties, exact identification of a type of a biological agent is essential.

3. Today’s Biological Agent Detection Equipment

(1) Trends in the Operational Concept

Many western countries divide biological agent detection devices into fixed facility equipment and field unit equipment. The equipment is issued to a chemical unit or a medical unit, and not to the other units or individuals yet.

a. Detection Equipment for a Fixed Facility (Fixed System)

Important national facilities, such as air bases, ports and communication centers, and public facilities such as airports, railroad stations, etc., where many people gather, are well known and located by enemies from peacetime. Such vulnerability of fixed facilities necessitates detection equipment more than combat units do. A fixed facility does not severely restrict size, weight, and energy consumption rate of an instrument, and a rather large one is allowed. And it must have high sensitivity and speed of identification rather than high resolution. This kind of equipment can be installed on a ship. A multi-point sensing system, as well as a stand-off instrument, is used for detecting air contamination of an area.

For an evacuation and treatment facility there is no severe restriction on size, weight, and energy consumption rate of an instrument, the instrument should have high resolution and sensitivity rather than rapid identification capability.

In order to detect water contamination a specific point-sensing instrument should be put in an open of a water supply system.

b. Detection Equipment for a Combat Unit (Field System)

There was no field system until recent date, thus a biological attack was detected by the epidemiological method based statistically on a sick book, and a biological agent was identified by the bioassay in which samples taken from patients were examined. It was unveiled that Aum-Shinrikyo cultured anthrax and Iraq had a secret biological program. In 1996 U.S. equipped the 310^th Chemical Company (Reserve) with M31 Biological Integrated Detection System which was the first practical field system in the world. The system is installed in a collectively protected S788 shelter, which is mounted on a high mobility multi-purpose wheeled vehicle (HMMWV). It is distributed to a chemical company (army reserve) that will be attached to an army corps. There is no other field instrument for combat units or individuals in the world.

(2) Technical Trends

Drugs and biological products are aggressively developed by means of a genetic engineering in the world. Medimmune Inc., Cephalon Inc., Genetech Co., Amgen Co., Alkaermes Inc., Human Genome Sciences Co., and Pfizer Inc. are famous genetic engineering companies in the U.S., and QLT Inc. in Canada. Those companies have a lot of good technologies that can be applied for biological detection. Biological agent detection technologies are classified into a microscopic method, a bioassay, a genetic analysis, and a physico-chemical method. They are also divided into a standoff or remote sensor, and a point sensor.

a. Standoff or Remote Sensor

A biological agent chemically comprises water, proteins, saccharides, lipids, etc. A delicate binding of the composition determines characteristics of an agent. Identification of an agent finally depends upon affirmation of its characteristics. Means of identifying characteristics of an agent are a microscopic method, a bioassay (an immunological method), a genetic analytic method. These methods cannot be applied to a stand-off or remote sensing in principle. The only method of remote sensing is a physico-chemical method that uses spectroscopy, but the method cannot determine characteristics of an agent, hence it is unable to identify an agent exactly. Remote sensing can be accomplished by electrically systemizing many point-sensors or by detecting only unusual event of most threatening aerosol without identification of an agent.

A sensing and tracking technology of an aerosol of 1-100μm in size, which is suitable to cause infection through a respiratory organ, is under development for an aerosol detecting device. When an aerosol is detected only by diameter of its particles, fog or other aerosols may be misconceived as a biological agent. Fortunately DNA and RNA have the characteristic absorption band at c.a. 260nm (ultraviolet), and a protein has it at c.a. 280nm (ultraviolet). The absorption bands can identify a biological agent.

b. Point Sensor

A microscopic method, a bioassay, a genetic analysis, and a physico-chemical method can be applied for a point sensor.

(a) Microscopic Method

This is the most traditional method, which uses a light-optic or an electron microscope. It is also one of the methods that can recognize characteristics of an agent. But it is not fit to detect and warn a biological attack. Reasons are that a light-optic microscope can only identify bacteria and fungi, and an electron microscope can only identify viruses. Additionally they cannot continuously monitor water and/or air, and they are not sensitive enough, and it takes time even for an expert to detect an agent by using them.

(b) Bioassay

This method uses a vital reaction to assay a biological agent quantitatively and qualitatively. The vital reaction includes inhibit of growth and sterilization by an antibiotics and/or an inhibitor, and identification of an agent includes a morphological or physiological biochemical characteristics. Conventional bioassays take several days or weeks to identify a bacterium. They also entail a risk of culturing a pathogen and the culture needs specific skill and a lot of labor. An environmental factor may kill the pathogen. Therefore the reliability of the assay is poor.

Recently, studies to detect an agent at a molecular level are under way. The studies carried out are a vital reaction between an agent and a fluorescence-marked cell or protein fixed on a silicon tip. The cell or protein reacts with an agent (such as an antigen-antibody reaction), and detects and identifies an agent. They are marked with a fluorescent or magnetic group or a radioisotope.

A fluorescent method is called ELISA (enzyme-linked immunosorbent assay), a magnetic method is called AFM (atomic force microscopy) and a radioactive method is called RIA (radioimmuno assay). When these technologies go into practical use, continuous monitoring water and air could be achieved, and portable field equipment would be practical.

The RFLP (restriction-fragment length polymorphism) analysis has been developed, in whose process a restriction enzyme cuts a specific part of a DNA and the DNA is identified by the electrophoresis pattern of the fragment－DNA fingerprint. The method is valuable in a laboratory, but it is not suitable for field use because it takes time to prepare and process a sample, and a gel is used for the electrophoresis apparatus. A new sensitive and rapid-detecting device is recently being developed, which uses a DNA probe that is a fragment of a single strand DNA marked with a radioisotope or a fluorescent group.

(d) Physico-Chemical Method

The simplest detector of an aerosol of a biological agent is a particle analyzer, which detects drifting particles in the air. It detects an agent very fast and continuously, but cannot identify an agent, furthermore all particles detected are not biological agents. It is, however, worth using as a primary sensor to turn a precise detector on.

A membrane of an agent comprises double layers of phospholipids, proteins and oligosaccharides, and others. The lipids and saccharides are unique to a microorganism respectively, therefore an agent can be identified by examining the molecular structure of them. They are higher polymers and then they cannot be analyzed directly by common physico-chemical analysis instruments. Recently some mass-spectrometer and chromatography for high polymers are developed. An example is a liquid- chromatography mass-spectrometer (LC-MS), in which a liquid-chromatography is directly connected in front of a mass-spectrometer and MALDI (Matrix-Associated Laser Desorption Ionization) or ESI (Electrospray Ionization) is used for destruction and ionization of polymers. And GLC (Gas-Liquefied Chromatography) is also developed.

These devices are very sensitive and able to identify an agent very fast and monitor water and air continuously, when an attached computer recognizes patterns of the output.

4. Progress in Sampling Technique and Its Effects

For a point detector, a sample containing a trace of an agent is often concentrated and processed properly. An aerosol sample may be concentrated by a centrifugal condenser or trapped by water. A trace of a DNA or RNA sampled is amplified rather than condensed. These sampling and processing methods have already reached completion. Followings are details of them.

(1) Centrifugal Concentration

This method uses a centrifugal force of a spiral airflow, which is caused by a whorled baffle plate put near an inlet. It has an advantage of attaining a concentrated sample in a short time.

(2) Trapping in Water

An aerosol is blown into water and the water traps and concentrates an agent. Sometimes filtration is necessary thereafter.

(3) Gene Amplification

The most popular gene amplification method is the Polymerase Chain Reaction (PCR) method, which was developed by a Swiss pharmaceutical company “Roche”. In the method a short nucleotide of a special purified DNA polymerase extracted from a thermophile and a chemically synthesized DNA (which is called “primary”) are used to clone a specific DNA sequence in vitro. They amplified DNA very fast; for example one billion times amplification takes only 2-3 hours. An automatic reaction apparatus is now sold in a commercial market. The reaction consists of 2 steps. At a first cycle an objective duplex DNA is extracted and the temperature of the DNA is raised to 95℃ in order to denature it and bind with the primer. The primer works as an initiator of the reaction. The DNA polymerase synthesizes the complimentary DNA at the coupling point of the primer and a duplex DNA is made. At a second cycle the synthesized duplex DNA is again heated and denatured to a single strand DNA, and a duplex DNA is synthesized again under the presence of the primer and the polymer. The cycles are repeated. The gene cloning method using a vector takes several days, whereas the PCR method takes only 1-2 hours.

Other gene amplification methods are developed recently. Examples are the TMA method developed by Geneglobe Inc. (US), the SDA method by Vectone-Dixon Inc. (US), the LAMP method by Eiken Chemical Ltd. (Japan), and the ICAN method disclosed September 2000 by Takara Breweries Ltd. (Japan).

Table-1 shows the characteristics of three typical gene amplification methods.

Table-1 Characteristics of Typical Gene Amplification Methods

	PCR	LAMP	ICAN
Production Company	Roche	Eiken Chemical Ltd.	Takara Breweries Ldt.
Template	DNA	DNA	DNA
Amplification Capacity	Middle	Large	Large
Temperature	60⇔95	ca. 60℃	ca. 65℃
Reagents	Not necessary	Not necessary	Necessary
Others	･The most popular method ･An automatic reaction apparatus is necessary.	･The temperature of the reaction is constant. ･Simple operation ･Low cost ･Restricted only for known short DNA’s	･The temperature of the reaction is constant. ･Simple operation ･Low cost ･Very high efficiency ･Applicable to a DNA chip

5. Japanese Technical Capability

Japan has enough capabilities to produce a physical portion of a biological detection device except for a LIDAR. However, Japan has a few physico-chemical and genetic data of a biological agent. Followings are details of Japanese capabilities.

(1) Physico-Chemical Analysis Technology

In Japan Shimazu Ltd. and many other companies produce many kinds of precise analysis instruments such as an electron microscope, a mass spectrometer, a liquid chromatograph and a gas chromatograph. These instruments are only for laboratory use, which is large in size and not hardened for field use, and consuming a large amount of energy. Moreover those companies have no database of the output to analyze a biological agent with them. Reduction of energy consumption and hardening of the instrument will be accomplished rather in short time, but making a database of biological agents will take time.

Some company recently made a diagnosis kit for influenza using a simple solid chromatograph, the company, therefore, has a production capability of a portable chromatograph.

NEC had once tried to manufacture a LIDAR for a remote sensing, but the equipment had not been fielded.

(2) Monoclonal Antibody Technology

A large amount of a purified antibody is needed to assay biologically. Takeda Pharmaceutical Inc., Teijin Ltd., Biomedical Institute, Dai-Nihon Pharmaceutical Inc., etc., produce many kinds of vaccines and antiviral agents on a commercial basis. Hence they may have enough capability to produce monoclonal antibodies.

(3) Electronics Technology

Japan is said to have high-level technologies of electronics, which is essential to reduce the size of equipment. Takara Breweries Ltd. is the leading company in the area of a DNA chip. But it takes time until a practical sensor with a DNA chip appears in the market.

(4) Software Technology

Specific software is necessary to collate instrumentally analyzed data with a database of biological agents, to identify the agent, and to warn contamination within predicted area. Aroka Ldt., Toshiba Ldt., Seiko EG&G Inc., etc., have software to analyze and identify radioisotopes rapidly from the output of a multi-channel energy analyzer for radioisotope-protection. It suggests that those companies could make a program, which identifies a biological agent by applying the radioisotope identification program, after a biological database is established.

6. Japanese Development of Biological agent Detection System

When Japan develops a biological agent detection system, Japan must decide how to develop it. The first choice is ①to develop a fixed system at first, then a field system, ②to develop a fixed system and a field system simultaneously, or ③to develop a field system directly. The second choice is use of ①a biological assay, ②a gene-analytic method, or ③a physico-chemical method.

With respect to the first choice, I intimate that Japan should develop a fixed system at first, thereafter a field system. The first reason is that a biological weapon is a strategic weapon and is also a terrorism weapon. Then the priority distribution of the system should be an important fixed facility. The GSDF has only developed field equipment before, but it should develop fixed equipment, too. The second one is that development of a fixed system is easier than a field system because the system has little limitation on energy consumption rate, weight, and size.

For the selection of a technical method we must consider detecting factors, sensitivity, response time, feasibility within 10-20 years, subsidiary effects of a development. Table-2 shows the characteristics of three technical methods. Important factors to decide are possibility of remote sensing, sensitivity, and response time. Up to the present time no technical method, which fulfills all of the important factors, is found. Even US, the most developed country in this area, uses a combination of various methods of detection: a particle analyzer to monitor continuously and a bioassay to identify an agent. I intimate that Japan should develop such combinatory system; the combination of a physico-chemical method, which has possibility of remote sensing, and a gene-analytic method, which advances rapidly.

Table-2 Comparison of three technical methods

	Biological Assay	Gene-Analytic Method	Physico-chemical Method
Detecting Factor	Identify agents at a point	Identify agents at a point	･Identify agents at a point ･Remote sensing of existence of agents
Continuous Monitoring	Possible, but no sign appears when an agent becomes absent	Possible, but no sign appears when an agent becomes absent	Possible
Sensitivity	Molecular level	Molecular level	Small quantity
Response Time	2～3 hours	1～2 hours	Several minutes
Feasibility	Relatively difficult	Relatively difficult	Relatively easy
Development Period	Middle term	Long term	Short term, but remote sensing takes long term
Cost	High	High	Low
Subsidiary Effects	･Applicable to a field system ･Capable of detecting a chemical agent ･Applicable to civilian use	･Applicable to a field system ･Applicable to civilian use	･Applicable to a field system ･Capable of detecting a chemical agent ･Applicable to civilian use

In the process of development, a database on structures and characteristics of agents at molecular level is essential. It is also valuable to develop a prophylactic and a remedy. US institutes are said to have already studied those of all Australian Group agents, and some of them are applied for patents. Development of a Japanese own detection system and/or protection device necessitates study on structures and characteristics of agents by Japan itself.

The other idea is to purchase the equipment from abroad. But development of the equipment enormously affects a pharmaceutical industry, a chemical industry, medicine. Thus I intimate that Japan should develop its indigenous system as a national project.

Afterword

The Defense Agency has discussed several times about protection against biological weapons at “the ad-hoc meeting of biological defense”. The final report was advanced to the Defense Ministry, recommending urgent establishment of detection and surveillance system. I hope that the system will be established soon, and Japan has enough capability to protect against biological weapons.