Gong Gong says

This is a posthumous blog of our father's (Lim Kok Ann) life. When our father passed away on 8 March 2003, he left behind an unpublished autobiography. We'd like to celebrate his life by sharing his autobiography through this blog.


"I have dredged these anecdotes from memory just to pass the time; if they amuse my grandchildren their purpose will have been served; if they provide any instruction, it will be a happy coincidence; that they are disjointed is probably to be expected.

Aurora was the name of my grandfather’s house in Kulangsu.   Amoy, where I spent the first five or six years of my life.   I still have vivid memories of events that took place when I was barely three years old.

Lim Kok Ann
October 1996"

Sunday, December 07, 2008

3:17 Invention of LBM antisera pools
Melnick’s laboratory was appointed by WHO as the International Enterovirus Centre that collaborated with national enterovirus centres in the identification of viruses isolated in polio outbreaks to determine what types of poliovirus were involved and if there were other enteroviruses that could cause polio-like symptoms (this was rarely the case).

(For convenience though not strictly correct, in the following account, the term enterovirus included those coxsackieviruses that grew in tissue culture. )

One important bit of collaborative work between different laboratories was the preparation and standardization of neutralizing or typing antisera. These were usually prepared by vaccinating rabbits or other animals with purified virus grown in tissue culture. It was most important that the virus used to make an antiserum should be of one virus type only, and complications sometimes resulted when a laboratory used a vaccine “virus” that had two different types of viruses in it. High quality typing antisera was as precious as gold because a rabbit does not yield much serum.

To match a virus isolate with one of 20 or more enterovirus (or coxsackievirus) types was no easy task. To avoid doing over twenty tests of which all but one was unnecessary, the identification test was done in steps. Three serum-pools were made, each containing a mixture of about one third of the typing sera that had to be typed against. If one of the serum-pools neutralized the virus isolate we proceeded to the next step, and that was to type the isolate against each component of the neutralizing serum-pool. This was relatively a simple procedure and I identified a number of virus isolates in this way.
One day it occurred to me that three serum-pools were used in step 1 because there were three poliovirus types, the commonest of which was poliovirus type 1. Serum-pool A contained neutralising antiserum for poliovirus type 1 and the antisera for a number of enteroviruses that were thought to be most common. Serum-pool B neutralized poliovirus type 2 and some other enteroviruses; Serum-pool C neutralized poliovirus type 3 and the remaining enteroviruses.

What if, I asked, I made up a Serum-pool D that contained some of the antisera that made up Serum-pool A, excluding poliovirus type 1 with some of the antisera that made up Serum-pool B, excluding poliovirustype 2. If a virus isolate was neutralised by Serum-pool D but not by Serum-pools A, B or C, it showed that the virus was not a poliovirus, but one of the “other” enterovirus types common to Serum-pools A and B that I had put in Serum-pool D.
Further thinking along this line led me to the realization that by making six antiserum-pools I could further narrow down the identification of a virus by its “neutralizing pattern” that corresponded to the way I put together the typing antisera. For example, if antiserum for each of six virus types was incorporated in only one of the six serum-pools, say Serum-pools A to F, and if each of the antisera for the remaining types were distributed in more than one pool, then neutralization by one serum-pool only showed that the virus isolate belonged to the type that was put in the serum-pool and none other! Moreover, with six serum-pools, I had available 15 double neutralization patterns, A+B, B+C, C+D and so on, and 20 triple (!) neutralization patterns, A+B+C, B+C+D, and so on, more than the number of existing enteroviruses. The principle will be familiar to those who bet in football pools and, of course, to those who played with permutations and combinations in algebra.

To recapitulate: I had hit upon the principle of identifying a virus isolate in one test, using antiserum pools in which the typing antisera had been distributed uniquely. If a virus was neutralized only by one Serum-pool A, B, or C..., it would be identified as enterovirus No.1, No.2 or No.3, as the case may be. If the virus was neutralized by two Serum-pools A and B, and by none other, it could be identified as enterovirus No.7, and if neutralized by Serum-pools A+C, as enterovirus No.8, and so on. The possibilities were limitless!

Melnick was intrigued by the labour- and time - saving aspects of my invention. The existing method required setting up three tests in the first step and possibly eight other tests against the individual components of a serum pool. If the identification could be achieved in one step, it halved the time necessary to identify a virus isolate. Melnick told me to concentrate on my bright idea.
There was quite a lot of work to be done to show that the combination pools system was workable. First, I had to find out if any enterovirus was neutralized by antiserum for other enteroviruses and to what extent. There were indeed one or two enteroviruses that gave such cross reactions. Then I had to choose the components of my serum-pools to take such cross-reactions into account. Eventually I had a set of six serum-pools that enabled the typing of the 24 types of enteroviruses (or coxsackieviruses) then known in Houston and I was ready to publish.
Mati played a great part in the work for she had made most of the typing antisera herself and she double-checked the validity of my experiments. The report was sent to the Journal of Immunology under the authorship of K.A.Lim and M. Benyesh-Melnick and entitled, Typing virus by Combinations of antiserum pools. Application to Typing of enteroviruses (Coxsackie and ECHO).

Note: ECHO viruses. When cytopathogenic agents were first discovered that were not poliomviruses, and apparently not related to any disease, they were called ‘orphan viruses’ , that is, viruses looking for diseases to be named after. Thus, the acronym for them, ECHO viruses: Enteric Cytopathogenic Human Orphan viruses -- ‘Human’ because similar viruses were found in monkeys.

Based on this pilot scheme I drafted a scheme for combining antisera for 42 enterovirus types in eight combination antiserum pools, but had to leave the details to Mati as my time at Baylor was up. WHO found my invention of practical value as Melnick had foreseen and Melnick was givena grant to make enterovirus antisera in large quantity and to make combination antiserum pools for distribution to national enterovirus centres. Melnick found that horses responded well to vaccination with enterovirus vaccines and gave antisera in two litre lots as compared with 20 ml for a rabbits. He freeze-dried the antiserum pools and distributed them under the label of LBM enterovirus antiserumpools (LBM for Lim &Benyesh-Melnick).
“We shall have enough material here to last well into the 21st century,” he told me. Whenever a polio outbreak takes place, the investigators take faecal specimens from cases and their contacts to determine the type of polio virus responsible for the outbreak and to try to trace the source. Many non-polio CPE agents were isolated, the great majority of them very likely enteroviruses. Rather than throw them away, attempts were made to identify them, and as few laboratories had a full range of enterovirus antisera, the “unknowns” were sent to the national public health laboratories from where they found their way to the regional WHO enterovirus laboratory.

The LBM antiserum pools freed regional centres from having to identify the unknowns usually sent to them. Instead, WHO distributed LBM antiserum pools to all laboratories working on polioviruses on the premise that those who can isolate poliovirus can do neutralization tests and identify any CPE agent believed to be enterovirus. Without the LBM antiserum pools it would have been necessary to distribute typing antiserum for each of the antisera that the antiserum pools comprised. In 1992 Melnick reported that the first batch of LBM antiserum pools he had made were exhausted and reviewed reports he had received concerning their use. He further stated that he was about to make a second batch of LBM antiserum pools from the horse antisera from which he had made the first batch, about half of which had been stored in deep freeze. He thus fulfilled the prediction he had made about the LBM pools being used in the 21st century.

Editor’s Note: Recently my nephew Koi Jun demonstrated a card trick, in which he was able to identify one card I had selected. He laid out combinations of cards and had me state which combinations contained my card. He was then able to identify my card ... using the same principle as his grandfather has described above for the LBM anti-sera typing pools.

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